Differentiating adult hippocampal stem cells into neural crest derivatives

Neuroscience 118 (2003) 1–5

LETTER TO NEUROSCIENCE DIFFERENTIATING ADULT HIPPOCAMPAL NEURAL CREST DERIVATIVES A. R. ALEXANIAN AND M. SIEBER-BLUM*

Abstract—To investigate the degree of plasticity of hippocampal neural stem cells from adult mice (mHNSC), we have analyzed their differentiation in co-culture with quail neural crest cells. In mixed culture, mHNSC give rise to several non-neuronal neural crest derivatives, including melanocytes, chondrocytes and smooth muscle cells. The data suggest that neural crest cell-derived short-range cues that are recognized across species can instruct adult mHNSC to differentiate into neural crest phenotypes. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. culture,

CELLS

INTO

response to stimuli from distinctly different cells that are derived from an unrelated species. To this end, mHNSC from adult ROSA26 mice (Friedrich and Soriano, 1991) were co-cultured with quail neural crest cells (qNCC). The neural crest is a transient embryonic tissue in vertebrates that originates in the neural folds and gives rise to a wide array of cell types and tissues of the adult organism (Le Douarin and Kalcheim, 1999). At the onset of migration, neural crest cells form a heterogeneous population that consists of stem cells, fate-restricted cells and committed cells (Sieber-Blum and Cohen, 1980; Sieber-Blum and Sieber, 1984; Sieber-Blum, 1989; Fraser and BronnerFraser, 1991; Stemple and Anderson, 1992; Wehrle-Haller and Weston, 1995). Neural crest cells generate various differentiated phenotypes in culture, including pigment cells, chondrocytes, and smooth muscle cells (Cohen and Konigsberg, 1975; Ito and Sieber-Blum, 1991; SieberBlum, 2000). When cultured alone, hippocampal NSC give rise to three phenotypes, neurons, astrocytes and oligodendrocytes (Fig. 1a, b) as described previously (Palmer et al., 1997). For co-culture, in vitro cloned adult mHNSC from ROSA26 mice were co-cultured with qNCC. mHNSC in co-culture, defined as Xgal-positive cells, expressed neural crest-derived phenotypes. In mixed culture, 19.1⫾3.2% of Xgal-positive mHNSC contained melanin granules (Fig. 1d–m; Table 1) and expressed MelEM (Fig. 1g–m) a marker for late progenitors in the melanogenic cell lineage (Nataf et al., 1993); 2.9⫾1.3% of mHNSC cells plated expressed collagen type II, a marker for chondrocytes (Fig. 1n–p), and 7.6⫾1.7% differentiated into smooth muscle cells (Fig. 1c; Table 1). Neither melanocytes nor chondrocytes differentiated in mHNSC culture in the absence of qNCC (Table 1). Equivalent results were obtained with mHNSC from 6- and 12-month-old mice. Rare Xgal-positive smooth muscle cells develop in mHNSC culture even in the absence of neural crest cells (Table 1), as described previously (Tsai and McKay, 2000). There is an inverse relationship between the number of cells plated and the percentage of mature smooth muscle cells developing in the absence of qNCC, indicating that this apparent default developmental pathway is counteracted by homotypic cell interactions and promoted by permissive culture conditions. The nuclei of qNCC and mouse hippocampal NSC have a distinctly different morphology, which is maintained in culture. In order to address possible fusion of mouse-

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA

Key words: ROSA26, quail, chondrocyte, smooth muscle cell.

STEM

melanocyte,

The goal of this work was to investigate the plasticity of neural stem cell from adult mouse hippocampus. Whereas neurogenesis ceases around birth in most areas of the brain, stem cells persist in the subventricular zone (SVZ) and hippocampal dentate gyrus and continue to generate neurons throughout adult life (Fuchs and Gould, 2000; Alvarez-Buylla et al., 1990; Goldman, 1995; Kuhn et al., 1996). Ciliated ependymal cells (Johansson et al., 1999), SVZ astrocytes as well as mature neurons can have neural stem cell (NSC) characteristics (Doetsch et al., 1999, Laywell et al., 2000; Alexanian and Nornes, 2001). Hippocampal NSC are located in the subgranule cell layer of the dentate gyrus (Kuhn et al., 1996; Eriksson et al., 1998). They proliferate in response to learning (Shors et al., 2001), physical exercise (Van Praag et al., 1999) and stress (McEwen, 1999). NSC isolated from the SVZ or from the entire forebrain are not irreversibly committed to their tissue of origin, but can under certain conditions be induced to form other cell types, including skeletal muscle (Galli et al., 2000), smooth muscle (Tsai and McKay, 2000) and hemopoietic cells (Bjornson et al., 1999). By contrast, the developmental potentials of hippocampal stem cells remain to be better defined. Hence, we sought to determine whether adult mouse hippocampal neural stem cells (mHNSC) could give rise to non-neuronal phenotypes in *Corresponding author. Tel: ⫹1-414-456-8465; fax: ⫹1-414-4566517. Abbreviations: DAPI, 4⬘,6-diaminidino-2-phenylindole dihydrochloride; mHNSC, mouse hippocampal neural stem cells; qNCC, quail neural crest cells; RT, room temperature; SVZ, subventricular zone.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(02)00994-6

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A. R. Alexanian and M. Sieber-Blum / Neuroscience 118 (2003) 1–5

Fig. 1.

A. R. Alexanian and M. Sieber-Blum / Neuroscience 118 (2003) 1–5

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Table 1. Quantitative analysis of differentiation of adult mHNSC into melanogenic, chondrogenic and myogenic cells in the presence and absence of qNCCa Cells plated (cells/cm2) mHNSC

Culture medium

Substrate

qNCC

Differentiated Xgal⫹ mHNSC (% of # cells plated⫾S.E.M.) Melanocytes

Chondrocytes

Smooth muscle cells

2000

None

100% NB/B27

Poly-D-lysine

0

0

0

1000

None

100% NB/B27

Poly-D-lysine

0

0

0

2000

None

50% NB/B27 50% C/␣-MEM

Poly-D-lysine plus collagen

0

0

0.9⫾0.2

1000

None

50% NB/B27 50% C/␣-MEM

Poly-D-lysine plus collagen

0

0

4.6⫾0.8

1000

3000

50% NB/B27 50% C/␣-MEM

Poly-D-lysine plus collagen

19.1⫾3.2

2.9⫾1.3

7.6⫾1.7

a mHNSC indicates mouse hippocampal neura stem cells; qNCC, quail neural crest cells; MC, melanocytes; NB/B27, neurobasal/B27 culture medium; C/␣-MEM (75% ␣-modified Minimal Essential Medium [MEM], 15% horse serum, 10% chick embryo extract; Laywell et al., 2000). As substrata poly-D-lysine plus collagen (1 ␮g/cm2 and 7 ␮g/cm2, respectively) or poly-D-lysine alone (10 ␮g/cm2) were used. The percent of smooth muscle cells was significantly increased in the presence of neural crest cells compared to pure mHNSC cultures (P⬍0.009).

derived mHNSC with quail-derived neural crest cells, or uptake by neural crest cells of ␤-galactosidase from dying mHNSC, we have determined the average nuclear area and average intensity of DAPI fluorescence in the three cell types (mHNSC, qNCC, Xgal-positive melanocytes; Table 2). The nuclear area of Xgal-positive melanogenic cells was similar to that of mHNSC cultured alone, identifying them as mouse cells. By contrast, the nuclear area of neural crest-derived melanocytes was significantly smaller (Table 2). These data strongly indicate that Xgal-positive melanocytes are derived from mHNSC, and that Xgal reactivity is not due to phagocytosis. The intensity of DAPI fluorescence was not significantly different in the three cell types (Table 2). This result excludes tetraploidy and thus cell fusion. Similar data were obtained with Xgal-positive chondrogenic cells. Our co-culture time is relatively short (7 days) and the frequency of Xgal-positive cells in coculture that express neural crest traits is relatively high (4.6-19.1%). While this does not preclude the occurrence of chromosomal transfer in our cultures, chromosomal transfer is unlikely to account for our data, as fusion processes occur at very low frequency (1 in 104 to 1 in 5⫻105) during prolonged co-culture (2– 4 weeks; Terada et al., 2002; Ying et al., 2002; Wurmser and Gage, 2002).

Our data extend current knowledge of adult mHNSC plasticity by demonstrating that mHNSC can generate several non-neuronal neural crest derivatives via neural crestderived cues that are not species-specific. All three cell types are characteristic neural crest derivatives, as the neural crest forms pigment cells of the skin and internal organs, craniofacial bone and cartilage, and smooth muscle of the cardiac outflow tract and proximal walls of the great vessels (Le Douarin and Kalcheim, 1999). None of the three cell types are generated within the brain. The nature of the instructive neural crest-derived signals remains to be determined. Three observations suggest that they have a short range of action, and may thus be conveyed by diffusible factors, direct cell– cell contact (Tsai and McKay, 2000) or cell fusion (Ying et al., 2002; Terada et al., 2002). First, the relative abundance of mouse mHNSC-derived pigment cells, chondrocytes and smooth muscle cells developing in co-culture mirror that of committed progenitor cells in neural crest cell culture (Sieber-Blum et al., 1993). Second, three different cell types develop within the same culture plate. Third, mousederived and quail-derived cells of the same phenotype were observed in close physical proximity. Our findings favor cell-to-cell signaling either by diffusible factors or

Fig. 1. Phenotypes generated in vitro by mouse hippocampal neural stem cells (mHNSC) cultured alone and co-cultured with quail neural crest cells (qNCC). (a– c) mHNSC culture. ␤-III tubulin immunoreactive neurons (Texas Red fluorescence), glial fibrillary acidic protein (GFAP) immunoreactive astrocytes (a, fluorescein fluorescence; b, Texas Red fluorescence), and O4-positive bipotent precursors (Palmer, 1997) and oligodendrocytes (b, fluorescein fluorescence) in clonal hippocampal cultures grown in defined culture medium (Neurobasal/B27). (c) In pure mHNSC culture, rare morphologically mature smooth muscle cells (Texas Red fluorescence) develop when grown in a 1:1 mixture of ␣-modified MEM (supplemented with 10% chick embryo extract and 15% horse serum) and Neurobasal/B27. Morphologically immature smooth muscle actin-immunoreactive cells were excluded from analysis. Melanocytes and chondrocytes were not observed. The mixed culture medium was chosen in order to accommodate the growth and differentiation requirements of both mHNSC and qNCC. (d–m) Melanogenesis in mHNSC/qNCC co-culture. (d) Xga-positive cell (arrow) contains melanin granules. Surrounding Xgal-negative melanocytes are of quail neural crest origin. (e) Corresponding DAPI nuclear stain. (f) Higher magnification of Xgal-positive melanocyte shown in (d). Both Xgal-reactivity (blue) and melanin granules (brown) are present in the same cell. (g–i) Colocalization of Xgal-reactivity and MelEM immunoreactivity (arrows) and DAPI nuclear stain at low magnification to show abundance of Xgal-positive MelEM-immunoreactive cells. (k–m) Higher magnification of cells marked by a square in (i). (k) Xgal-positive cell (arrow) contains a few melanin granules and expresses the melanoycte marker, MelEM (l, arrow). (m) DAPI nuclear stain of same field. (k, m) A neighboring quail neural crest-derived pigment cell is visible in close proximity. Note the difference in nuclear sizes. (n–p) Chondrogenesis in mouse mHNSC/qNCC co-culture. (n) Xgal-positive mouse mHNSC-derived cell (arrow) expresses collagen type II (o; arrow), a marker for chondrocytes. (p) Corresponding DAPI nuclear stain. Scale bars (a– c)⫽100 ␮m; (d, e)⫽100 ␮m, (f)⫽25 ␮m; (g–i)⫽250 ␮m; (k–m)⫽50 ␮m; (n–p)⫽50 ␮m.

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Table 2. Nuclear area and DAPI fluorescence intensity in Xgal-positive melanogenic cells, neural crest-derived pigment cells and mHNSC Cell type

Average nuclear area⫾S.E.M. (␮m2)

Average fluorescence intensity⫾S.E.M. (gray scale)

Xgal-positive melanogenic cells mHNSC Neural crest-derived melanocytes

139.2⫾8.9 153.5⫾7.6 47.7⫾1.9

105.2⫾1.6 105.8⫾0.8 99.2⫾3.5

The average nuclear area of Xgal-positive melanogenic cells and mouse hippocampal neural stem cells (mHNSC) was not significantly different (P⫽0.12). By contrast, the average nuclear area of neural crest-derived, Xgal-negative melanocytes was significantly different from that of Xgal-positive melanogenic cells (P⫽0.0001) and of mHNSC (P⫽0.0001). The average DAPI fluorescence intensity of Xgal-positive melanogenic cells was not significantly different from either neural crest-derived pigment cells (P⫽0.14) or mHNSC (P⫽0.88).

direct cell contact, whereas cell fusion or chromosome transfer appears less likely.

EXPERIMENTAL PROCEDURES All experiments were performed according to national and international guidelines on the ethical use of animals, using the smallest possible number of animals and minimizing suffering. Hippocampi of 6 – 8-week-old heterozygous ROSA26 mice were dissected, minced, dissociated (0.05% papain, 30 min at room temperature; RT), and pooled supernatants rinsed by centrifugation. The pellet was resuspended in NeurobasalA/B27 with 0.5-mM glutamine and plated at 250 cells/mm2 in 3.5-mm plastic dishes that were coated with poly-D-ornithine. After 1 h, unattached cells and debris were removed and the cultures incubated in Neurobasal/B27 medium supplemented with 0.5-mM glutamine, 50 units/ml penicillin, 50 ␮g/ml streptomycin and 20 ng/ml FGF-2. Half of the culture medium was replaced every 3 days with fresh medium. To clone mHNSC, cultures at confluence were trypsinized shortly. Foci of proliferating cells, located usually at the top of the multilayered culture, were detached, dissociated mechanically into a single-cell suspension, and replated at 100 cells per 3.5-cm plate in Neurobasal/B27 medium that was mixed 1:1 with medium conditioned for 24 h by cells in high-density hippocampal culture. Single cells were marked. After 2–3 weeks, single cell-derived clusters of proliferative cells were harvested using glass cloning rings and used for mHNSC/qNCC co-cultures. Neural crest cell primary explants were prepared exactly as described (Sieber-Blum, 2000). For secondary culture, cells were resuspended by trypsinization, diluted to 5000 cell/ml and plated at 1 ml per plate in collagen-coated 35-mm plates for 3 days. Three-day-old neural crest cell secondary cultures were resuspended and used for co-culture. For co-culture, cloned neural stem cells and neural crest cells were mixed at a ratio of 1:3 and grown on poly-D-lysine/collagen-coated glass coverslips for 7 days in 50% Alpha-MEM⫹50% Neurobasal A/B27, supplemented with 7.5% of a selected batch of horse serum and 5% day–11 chick embryo extract, to accommodate the growth and differentiation of cells from both sources. For combined X-gal cytochemistry and indirect immunocytochemistry, day-6 co-cultures were fixed with 4% paraformaldehyde in PBS (20 min at RT), and processed for Xgal histochemistry according to Tanaka et al. (1999). The next day, the cells were permeabilized (7 min; 2% normal goat serum, 1% BSA and 0.5% Triton X-100 in CMF–PBS), and incubated for 1 h with one of the following primary antibodies in the same solution (except for

10-fold less Triton X-100); rabbit polyclonal anti-␤-III-tubulin antibodies (1:400; gift of A Frankfurter; Katsetos et al., 1994); monoclonal (1:80) or rabbit polyclonal (1:100) anti-glial fibrillary acidic protein antibodies (Sigma, St. Louis, MO, USA); monoclonal antibody to ␣-smooth muscle actin, (1:350; Sigma, St. Louis, MO, USA); and antibody to oligodendrocyte O4, (1:100, Chemicon, Temecula, CA, USA). Immunoreactive cells were visualized with Texas Red-conjugated goat anti-mouse IgG or fluorescein-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Nuclear area and intensity of DAPI fluorescence were measured with Metamorph software (Universal Imaging Corp., Downingtown, PA, USA). Acknowledgements—This work was supported by USPHS Grant NS38500 (M.S.B.).

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(Accepted 19 November 2002)