A neurosphere-derived factor, cystatin C, supports differentiation of ES cells into neural stem cells

Takeo Kato*, Toshio Heike*†, Katsuya Okawa‡, Munetada Haruyama*, Kazuhiro Shiraishi*, Momoko Yoshimoto*, Masako Nagato*, Minoru Shibata*, Tomohiro Kumada*, Yasunari Yamanaka*, Haruo Hattori*, and Tatsutoshi Nakahata*

*Department of Pediatrics and ‡Biomolecular Characterization Unit, Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan

Communicated by Tasuku Honjo, Kyoto University, Kyoto, Japan, November 15, 2005 (received for review July 21, 2005) Although embryonic stem (ES) cells are capable of unlimited tatin C. Our results have led to the discovery of a previously proliferation and pluripotent differentiation, effective preparation uncharacterized biological activity of cystatin C, which induces of neural stem cells from ES cells are not achieved. Here, we have NSCs from ES cells exclusively. directly generated under the coculture with dissociated primary neurosphere cells in serum-free medium and the same effect was Results observed when ES cells were cultured with conditioned medium of ES Cells Differentiate into Neural Stem Cells by Coculture with primary neurosphere culture (CMPNC). ES-neural stem cells (NSCs) Dissociated Primary Neurosphere Cells. To determine whether NSCs could proliferate for more than seven times and differentiate into can regulate the differentiation of ES cells into neural lineages, D3 , , and in vitro and in vivo. The ES cells, which constitutively express GFP, were cocultured in responsible molecule in CMPNC was confirmed by matrix-assisted suspension with dissociated primary neurosphere cells for 21 days, laser desorption͞ionization time-of-flight mass spectrometry, and phenotypical changes in the GFP-positive ES cells were eval- which turned out to be cystatin C. Purified cystatin C in place of the uated. After coculture for 21 days, round spheres measuring Ͼ100 CMPNC could generate ES-NSCs efficiently with self-renewal and ␮m in diameter could be generated, which were expressed GFP multidifferentiation potentials. These results reveal the validity of (Fig. 1A). The number of ES cell-derived GFP positive spheres cystatin C for generating NSCs from ES cells. generated from 105 ES cells was 476.67 Ϯ 32.87, by coculture with dissociated primary neurosphere cells (Fig. 1B). When ES cells coculture system ͉ conditioned medium of primary neurosphere culture were cultured alone, the majority of ES cells died and only a few small cell clusters were generated (21.25 Ϯ 9.50 generated from 105 eurogenesis is considered to be the most complex event of ES cells). Thus, ES cells could generate spheres efficiently during Norganogenesis during embryonic development and involves a coculture with dissociated primary neurosphere cells in the pres- precise signaling, along with cellular interaction cascade, to gener- ence of FGF2 and EGF. Selectively FACS-sorted ES cell-derived ate the functional cellular networks. The embryonic organizer GFP-positive cells could proliferate and again formed spheres in a allows cells in its vicinity to execute their default neural program by defined serum-free medium with FGF2 and EGF. To exclude the emitting bone morphogenetic protein antagonists (1, 2). However, possibility of cell fusion (16) between ES cells and primary neuro- other works suggest a more complex mechanism (3–5). sphere cells, we carried out FACS analysis for DNA contents of ES Neural stem cells (NSCs) are the self-renewal, multipotent cells cell-derived cells. All ES-derived GFP-positive cells were diploid that generate neurons, astrocytes, and oligodendrocytes (6, 7). They and, thus, suggested that no cell fusion occurred in this coculture have great potential as a therapeutic tool for the repair of a number system (data not shown). of (CNS) disorders. Several in vitro systems When individual spheres were encouraged to fully differentiate, allowing derivation of neuronal progeny from embryonic stem (ES) cells positive for the neuronal markers microtubule-associated cells, which differentiate into all of the cell fates in a developing protein 2 (MAP2) and ␤-tubulin type III (Tuj) appeared 3 days embryo, have been described. However, attempts to exclusively after differentiation (Fig. 1C). Subsequently, cells positive for the generate NSCs or neural progenitor cells from ES cells are re- marker glial fibrillary acidic protein (GFAP) (Fig. 1D) stricted. It has been shown that neural fates emerge from ES cells and marker Gal C (Fig. 1E) could be detected in the serum-free conditions (8, 9). Although these procedures are after 7 days. The ES cell-derived spheres could be propagated noteworthy, both the quality and the quantity of ES-derived neural repeatedly, at least 7 times (Fig. 1I). Notably, the spheres retained cells are not sufficient for further examination or clinical applica- multilineage potential (Fig. 1 F–H). To examine whether the tions. Higher levels of neural differentiation are achieved by characters of ES-derived neurospheres changed with passaging, treatment of embryoid bodies with retinoic acid (RA) in the gene expression profiles of ES cell-derived spheres were examined presence of FCS (10, 11) or by coculture with particular stromal cell by RT-PCR. Nestin, which is expressed in NSC in vivo, the neuronal lines (12, 13). The action of RA is pleiotropic (14, 15), whereas the marker Tuj, the astrocytic marker GFAP, and the oligodendrocyte effect of several stromal cell lines is attributed to an undefined marker Gal C were expressed consistently during passaging (Fig. neural inducing activity. These factors severely restrict the ability to 1J). These results suggested that ES cells differentiated into NSCs, use cells cultured with FCS or with particular stromal feeder cells which had both self-renewal and multilineage differentiation po- tentials during cocluture with dissociated neurosphere cells.

for therapeutic treatments. NEUROSCIENCE Here, we developed an efficient system for the generation of ES cell-derived NSCs (ES-NSCs) during coculture with dissociated neurosphere cells without a need for FCS or feeder cells, which Conflict of interest statement: No conflicts declared. creates a bottleneck for therapeutic methods. We demonstrated Abbreviations: ChAT, cholinergic marker anti-acetylcholine transferase; CMPNC, conditioned medium of primary neurosphere culture; DBH, dopamine-␤-dehydroxyrase; that neurosphere-derived factor (NDF), which induces the gener- ES-NSC, ES cell-derived neural ; GFAP, glial fibrillary acidic protein; MAP2, micro- ation of ES cell-derived neurospheres, exists in the conditioned tubule-associated protein 2; MBP, myelin basic protein; NDF, neurosphere-derived factor; medium of primary neurosphere culture (CMPNC). We charac- NSC, ; TH, tyrosine hydroxyrase; Tuj, ␤-tubulin type III. terized this NDF by using chromatography and mass spectrometric †To whom correspondence should be addressed. E-mail: [email protected]. identification, revealing that this activity derives mainly from cys- © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0509789103 PNAS ͉ April 11, 2006 ͉ vol. 103 ͉ no. 15 ͉ 6019–6024 Downloaded by guest on September 26, 2021 Fig. 2. Generation of ES cell-derived neurospheres by using CMPNC. The addition of CMPNC increased the number of ES cell-derived spheres (A) and total cells (B). Data are means Ϯ SD of triplicate determinations from two or three independent experiments.

ical analysis revealed that the majority of cells derived from CMPNC-treated ES cell-derived spheres were nestin-positive (Fig. 3A). When individual spheres were encouraged to fully differenti- ate, Tuj- or MAP2-positive cells were detected at day 3 (Fig. 3 B and C). Subsequently, cells positive for GFAP, galactocerebroside, and myelin basic protein (MBP) appeared at day 7 (Fig. 3 D–F). When we evaluated the characteristics of Tuj-positive cells differentiated from ES cell-derived neurospheres, four neuronal subtype markers were detected: the dopaminergic neuron markers tyrosine hy- Fig. 1. ES cells differentiate into NSCs by coculture with dissociated primary droxyrase (TH) and anti-dopamine transporter, cholinergic neuron neurosphere cells. (A) ES cells, which constitutively express GFP, cocultured with marker anti-acetylcholine transferase (ChAT), serotonergic neuron dissociated primary neurosphere cells form spheres in the presence of FGF2 (10 ͞ ͞ ␮ marker serotonin, or norepinephrine͞epinephirine neuron marker ng ml) and EGF (20 ng ml) after 21 days (Scale bars: 100 m.) (B) ES cells could ␤ generate spheres efficiently (filled bar). However, when ES cells were cultured dopamine- -dehydroxyrase (DBH) were detected (Fig. 3 G–K). alone, the majority of ES cells died and only a few small cell clusters were Among them, 75–90.9% of ES cell-derived neurospheres contained generated (open bar). Data are means ϮSD of triplicate determinations from two TH-positive cells during the differentiation for 7 to 14 days (Fig. or three independent experiments. (C–H) Differentiated ES-derived spheres con- 3P). This value was much higher than that for cholinergic or tain neurons (C, MAP2), astrocytes (D, GFAP), and oligodendrocytes (E, galacto- serotonergic neurons (ChAT: 45%, 7 days; 55%, 14 days; and cerebroside). ES-derived spheres passaged five times retained also multilineage serotonin: 12%, 7 days; 25%, 14 days, respectively). To further potential (F, Tuj; G, MAP2; H, MBP). (Scale bar: 50 ␮m.) (I) ES-derived spheres could be served to the repeated propagation. (J) The expression of Nestin, Tuj, GFAP, confirm the preferential generation of dopaminergic neurons, we and Gal C genes in primary, twice-passaged, and four times-passaged ES-derived examined the expression of the mesencephalic dopaminergic neu- spheres was determined by RT-PCR. Lanes: 1, negative control; 2, primary ES- ron markers Nurr1 and En1. These markers were presented in ES derived spheres; 3, P2, twice-passaged ES-derived spheres; 4, P4, four times- cell-derived neurospheres induced by CMPNC (Fig. 6). These data passaged ES-derived spheres. suggested that ES cell-derived neurospheres could differentiate into neurons with various kinds of , predomi- nantly into dopaminergic neurons. Generation of ES Cell-Derived Neurospheres by Using a Secreted The ES cell-derived neurospheres induced by CMPNC could be Factor from Primary Neurosphere Culture. The biological activity that propagated repeatedly (Fig. 3Q), at least nine times, without losing induced the differentiation of ES cells into multipotential NSCs either multilineage differentiation activity or their preference to- during coculture with dissociated primary neurosphere cells could ward dopaminergic lineage (see Fig. 7, which is published as derive from two possible sources: soluble factors secreted from supporting information on the PNAS web site). To confirm whether primary neurospheres or direct cell-to-cell contact molecules. To a single ES-NSC can form a sphere in response to CMPNC evaluate the contribution of soluble factor secreted from primary neurospheres, we examined whether ES cell-derived neurospheres treatment, single-cell suspension cultures were prepared. Spheres could be induced in the presence of CMPNC. Addition of the could be detected at day 12 with the increasing cells in number CMPNC to the ES culture significantly increased the total numbers during culture (Fig. 3R). However the overall sphere formation of both cells and spheres (Fig. 2). Serial dilution of CMPNC at least value of single ES-NSC was less than that of NSC from embryos, up to 100 times could preserve this activity. because the majority of ES cells failed to generate spheres and To evaluate the gene expression profiles of ES cell-derived simply died (Table 1, which is published as supporting information neurospheres, expression of genes restricted to neural or nonneural on the PNAS web site). Nevertheless, these spheres that did form lineages were examined by RT-PCR (Fig. 6, which is published as retained multilineage potential. These results imply that NDF, supporting information on the PNAS web site). In ES-derived which induces the generation of ES cell-derived neurospheres, neurospheres formed by CMPNC treatment, expression of Rex1, exists in the CMPNC. which is highly expressed by inner cell mass of blastcytes and by ES We next explored the differentiation capability of ES cell-derived cells (17), was not detected. Simultaneously, Oct4, which is ex- neurospheres in vivo. Dissociated ES cell-derived neurosphere cells pressed in ES cells and primitive ectoderm cells (17), was down- expressing the GFP marker were injected into the hemiventricular regulated. In contrast, the expression of nestin, OTX2, and Mash1 area of neonatal mice. ES cell-derived cells could be detected by genes, all of which are expressed in NSCs in vivo, were confirmed. GFP expression in six of eight mice at 4 weeks and seven of eight Furthermore, multilineage neural markers, Tuj, GFAP, and Gal C, mice at 8 weeks by visual observation (Fig. 3M). In serial sections, were expressed in ES-derived neurospheres. Nonneural lineage TH-positive cells with GFP expression could be detected around genes, including the endodermal markers GATA4 and HNF4a (18, the (Fig. 3N). GFP-positive cells with GFAP 19), the mesoderm marker brachyury (20), and epidermal marker expression could also be detected (Fig. 3O). On the other hand, CK17 (21), were not detected. These gene expression profiles teratoma-like structures or reduced lifespans were not observed. observed in ES-derived neurosphere cells were comparable to those These results imply that ES cell-derived neurosphere cells could observed in primary neurospheres. Moreover, immunocytochem- differentiate in vivo without leading to tumor formation.

6020 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0509789103 Kato et al. Downloaded by guest on September 26, 2021 we evaluated whether cystatin C could replace CMPNC in inducing ES cells to differentiate into NSCs. To evaluate the generation of ES-NSCs, ES cells were cultured in suspension in the presence of FGF2 and EGF for 21 days with or without cystatin C. Addition of recombinant mouse cystatin C (R & D Systems, 1238-PI) at various concentrations (20 pg͞ml to 200 ng͞ml) significantly increased the number of total spheres generated from ES cell cultures (Fig. 4A). Few ES cell-derived spheres were generated in the absence of cystatin C. The efficiency of sphere formation by using recombinant cystatin C was Ϸ80% compared to that using conditioned medium. In the presence of cystatin C, addition of either FGF2 or EGF significantly increased the number of ES cell-derived spheres (Fig. 4B). Interestingly, the addition of cystatin C alone increased number of spheres, as compared with the number of spheres generated in the presence of EGF and FGF2 (P Ͻ 0.05). Further- more, even in the presence of either FGF2 or EGF, or both at a high concentration, the number of spheres was not increased (data not shown). The expression of cystatin C was detected in primary neurospheres by RT-PCR, but not in undifferentiated ES cells. Furthermore, Western blotting analysis revealed cystatin C was contained in the CMNPC (data not shown). Therefore, we spec- ulated that the main source of NDF activity in CMPNC is derived from cystatin C. Fig. 3. CMPNC-treated ES-derived spheres had multilineage differentiation potential and self-renewal potential. Differentiated ES-derived spheres contain When individual cystatin C-treated ES cell-derived spheres were nestin-positive cells (A, Cy3-nestin). ES-derived spheres could differentiate into cultured in the differentiation culture conditions, each of the neurons (B and D, Cy3-Tuj; C, MAP2), astrocytes (D and E, FITC-GFAP), oligoden- differentiated spheres contained MAP2-positive or Tuj-positive drocytes (E, Cy3-MBP; F, Cy3-Gal C). They could differentiated into various ma- cells at day 3 (Fig. 4D). GFAP-positive cells and MBP-positive cells tured neurons (G, Cy3-TH; H, Cy3-DAT͞FITC-Tuj; I, Cy3-ChAT; J, Cys-3-DBH; K, were detected at day 7 (Fig. 4 D and E). Furthermore, immuno- Cy3-Serotonin). Transplantation of ES cell-derived neurospheres into the neona- cytochemical staining for subtype-specific neuronal markers re- tal mouse brain. All grafts were easily detected by the GFP expression as ES- vealed that four neuronal subsets, dopaminergic, cholinergic, sero- derived cells (M) as compared with control (L). In serial sections, TH-positive cells tonergic, and adrenergic, were present in the Tuj-positive cell (Fig. (N, Cy3-TH) and GFAP-positive cells (O, Cy3-GFAP) with GFP expression could be 4 F–I). Moreover, cystatin C-treated ES cell-derived spheres could detected around the subventricular zone. (P) A time course study of the appear- ance of ES-derived neurospheres with Tuj, TH, DBH, and serotonin-like immuno- propagate at least seven times (Fig. 4C) without losing either reactivities. Positive sphere was defined as a sphere containing 10 or more cells multilineage differentiation activity or their preferred differentia- with immunoreactivity of each antibody. (Q) ES-derived spheres could be served tion toward dopaminergic neurons (see Fig. 9, which is published as to the repeated propagation in the presence of FGF2 and EGF. (R) Single-cell supporting information on the PNAS web site). To further confirm suspension culture in serum-free medium containing 0.3% agar were prepared. the presence of dopaminergic neurons, we examined dopamine Sequential images of a single CMPNC-treated ES-derived cell at 1.5, 4, and 12 days production in the induced neurons by reverse-phase HPLC. In after culturing. (Scale bars: 50 ␮m.) response to a depolarizing stimulus (56 mM Kϩ), ES cell-derived neurons released a significant amount of dopamine into the me- dium (Fig. 4J). These results indicate that functional neurons Purification and Characterization of NDF. Next, we evaluated the producing dopamine were generated with this method, suggesting physiological characteristics of NDF in CMPNC from the viewpoint that the ES cell-derived spheres generated by cystatin C are indeed of heat instability and molecular size. Heat treatment of the neurospheres. CMPNC at 60°C for 30 min or 100°C for 10 min abolished its activity. This biochemical characteristic strongly suggested that Cystatin C Regulates the Commitment of ES Cell Differentiation into NDF is protein-based. Further investigation to determine the Neural Lineage. Cystatin C could regulate neurosphere generation molecular mass by using an ultrafiltration procedure demonstrated either by direct induction of ES cells into NSCs or by expansion of that NDF activity was recovered in the 3,000–20,000 Da molecular cells that had spontaneously differentiated into NSCs. As shown in mass fraction. To purify the NDF, large quantities of CMPNC were Fig. 4B, a small number of small cell clusters could be generated generated (2 liters). After concentrating conditioned medium even in the absence of any growth factors. To address whether 200-fold by ultrafiltration, the NDF was semipurified through cystatin C induced NSC differentiation de novo or merely expanded phenyl hydrophobic column by step gradient elution with decreas- cells that were already committed to a neural lineage, we compared ing concentration of ammonium sulfate from 1 M to 0 M. The NDF the characteristics of spheres and small cell clusters generated in the activity was detected in the fraction eluted at the0Mconcentration presence or absence of cystatin C and in the absence of EGF and of ammonium sulfate. When this fraction was analyzed by SDS͞ FGF2. Immunocytochemical analysis revealed that the majority of PAGE, seven distinct bands were detected at the range from 3,000 cells derived from cystatin C-treated ES cell-derived spheres were to 20,000 Da molecular mass range (Fig. 8A, which is published as nestin-positive and that many more Tuj-positive cells were present

supporting information on the PNAS web site). After trypsin in spheres that developed in response to cystatin C. In contrast, only NEUROSCIENCE treating excised gel bands, proteins were identified by MALDI- a few nestin-positive cells were detected within the small cell TOF͞MS peptide mapping. Bands 1–3, 4, and 7 matched similar to clusters generated in the absence of cystatin C, and neither GFAP- peptidylprolyl isomerase A, mouse cystatin C (Fig. 8 B and C) and nor MBP-positive cells were detected after differentiation (Fig. 5 mouse profilin2, and insulin, respectively. A–E). Furthermore, small cell clusters could not reform spheres, and they simply died in the single cell culture system. Thus, they had Cystatin C Confers the NDF Activity. Recently, it was demonstrated neither self-renewal nor multilineage differentiation potentials. that the proliferation of NSCs in vitro and neurogenesis in vivo are To define the characteristics of cystatin C-treated ES cell-derived stimulated by the cooperation between FGF2 and cystatin C (21). spheres and spontaneously arising small spheres, we compared the Considering the close correlation of cystatin C with neurogenesis, expression of several transcription factors by RT-PCR (Fig. 5F).

Kato et al. PNAS ͉ April 11, 2006 ͉ vol. 103 ͉ no. 15 ͉ 6021 Downloaded by guest on September 26, 2021 Fig. 4. Cystatin C exercised the NDF activity, which promote to differentiate into NSCs from ES cells. (A) The addition of the recombinant mouse cystatin C increased the number of ES cell-derived spheres. The filled bars indicated the number of spheres, and the open bars indicated the number of small cell clusters. (B) Growth factor dependency of forming ES-derived spheres. In the presence of cystatin C, addition of either FGF2 or EGF significantly increased the number of ES cell-derived spheres. Filled bars indicate the number of spheres, and the open bars indicate the number of small cell clusters. (C) ES-derived spheres could be served to the repeated propagation over seven times and had multilineage differentiation activity (D, Cy3-Tuj͞FITC-GFAP; E, Cy3-MBP). They also could differentiated into various matured neurons (F, Cy3-TH; G, Cy3-ChAT; H, Cy3-Serotonin; I, Cys-3-DBH). (Scale bars: 50 ␮m.) (J) Example of an HPLC chromatogram showing high levels of dopamine (DA) in the medium of ES-derived neurons (Upper, green line, medium conditioned for 24 h). Relatively low basal DA release was detected (blue line, exposure to buffer for 15 min), as compared to the high levels of DA after 15 min of KCL-evoked depolarization (Lower, red line). Data are means Ϯ SD of triplicate determinations from two or three independent experiments.

Although nestin was expressed in the both spheres, high levels of spheres formed under these two different culture conditions are En1 and Nurr1, which control differentiation of dopaminergic and phenotypically distinct and that NSCs, which have both self-renewal serotonergic neurons in the midbrain and hindbrain (22, 23), were and multilineage differentiation potentials, could be generated in expressed only in cell populations treated with cystatin C. Rex1 was the presence of cystatin C. This result suggests that cystatin C expressed in untreated spheres, but was down-regulated in cystatin regulates the commitment of ES cell differentiation into NSCs. C-treated spheres. Brachyury, which is abundant in EBs, was not expressed in both (data not shown). These results demonstrate that Discussion In this paper, we reported an efficient system for in vitro NSC induction from mouse ES cells in the presence of cystatin C. First, we showed that NSCs could be generated from ES cells by coculturing them with dissociated primary neurosphere cells. We also showed that when CMPNC was added into the serum-free culture of ES cells, they generated the neurospheres. These results suggested that soluble NDF had an active role in the generation of ES-NSCs. Finally, we then have purified cystatin C in CMPNC as the NDF activity. Cystatin C, a cysteine protease inhibitor (24), is a molecule with pleiotropic functions (25, 26). It is a 14-kDa protein (120 amino acids) and is secreted by different cell types in vitro (27). Several proteinase inhibitors, such as thrombin inhibitors, calpain inhibi- tors, or cysteine protease inhibitors, have been suggested to be part of a regulatory system associated with neuronal differentiation. We showed that ES cell derived-neurospheres induced by cystatin C were nestin-positive and could differentiate into neurons and both in vitro and in vivo and had self-renewal potential. The Fig. 5. Cystatin C supported to differentiate into NSCs from ES cells. (A and expression profiles of several genes, including Rex1, Oct4, Otx2, B) Immunocytochemical analysis of nestin-positive cells constituted small cell nestin, and mash1, in cystatin C-treated ES cell-derived neuro- clusters and spheres, which developed either in the absence or presence of spheres were comparable to those in primary neurospheres. Fur- cystatin C. (C and D) Significantly more Tuj-positive cells differentiated from thermore, nonneural genes, including GATA4, HNF4, brachyury, spheres developed in the presence of cystatin C than small cell clusters devel- and CK17, were not expressed in either the CMPNC or in cystatin oped in the absence of cystatin C. (Scale bars: 100 ␮m.) (E) Immunocytochem- C-treated ES-NSCs. Previous studies have reported the differen- ical analysis of Tuj-, GFAP-, and MBP-positive cells differentiated from spheres tiation of NSCs from ES cells. Tropepe et al. (9) reported the developed in the presence of cystatin C and small cell clusters developed in the Ϯ induction of a small number of primitive neural stem cells from ES absence of cystatin C at day 3 and day 7. Data are means SD of triplicate cells in the presence of leukemia inhibitory factor and FGF2, determinations from two or three independent experiments. (F) Comparisons were made about the expression of Rex1, nestin, Nurr1, and En1 in the cystatin although these cells expressed an endodermal marker GATA4 in C-treated and cystatin C-untreated ES-derived sphere by RT-PCR. Lanes: 1, vitro and had neural and nonneural lineage potentials in vivo. negative control; 2, ES cells; 3, primary neurospheres; 4, cystatin C-treated ES Furthermore, in our laboratory, primitive NSCs could not be cell-derived spheres; 5, cystatin C untreated ES cell-derived small cell clusters. propagated repeated more than three times. We conclude that the

6022 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0509789103 Kato et al. Downloaded by guest on September 26, 2021 characteristics of cystatin C-treated ES-NSCs in this culture system and differentiated into dopamine neurons. Moreover, no tera- are analogous to those of NSCs. However, the efficiency of sphere toma-like structures were observed in any of the 16 mice that formation by using recombinant cystatin C was only Ϸ80% of that received grafts. Tumor formation is a major problem associated using CMPNC. This result suggests that NDFs other than cystatin with ES cell-derived cell grafting in the treatment of neurological C may play a role for neural induction of ES cells. diseases. Recently, it was demonstrated that the proliferation of NSCs Here, we have developed an efficient system for the generation in vitro and neurogenesis in vivo are both stimulated coopera- of ES-NSCs by using the soluble factor, cystatin C, without a need tively by FGF2 and cystatin C (28). This report infers that for FCS or feeder cells, which creates a bottleneck for therapeutic cystatin C plays a role in supporting the propagation of cells that methods. Furthermore, cystatin C-treated ES cell-derived cells already have differentiated into NSCs. Here, we demonstrated could proliferate continuously while maintaining a high differen- that the characteristics of spheres generated in the absence or tiation activity during continuous culture. Thus, we believe that presence of cystatin C are qualitatively different, and NSCs, cystatin C-treated ES-NSCs provide a good and safe cell source for which had both self-renewal and multilineage neural differen- CNS transplantation therapy, and this system provides a good tiation potentials, could be generated only in the presence of model to investigate the mechanism of CNS development and to cystatin C (Fig. 5 A–E). Therefore, we concluded that cystatin C realize many of potential applications in neuroscience and regen- had a previously uncharacterized function of regulating the erative medicine in the CNS. commitment of ES cells differentiation into NSCs, either directly or indirectly, in addition to its ability to stimulate propagation of Materials and Methods the cells that have already differentiated into NSCs. ES Cell Lines. The ES cell lines used in this study were the CCE cell How does cystatin C induce neural differentiation of ES cells? line and the D3 cell line with transfected green fluorescent protein We demonstrated that addition of cystatin C alone increased the (GFP) gene driven by the ubiquitous CAG promoter. Undifferen- number of spheres in serum-free conditions but that high concen- tiated ES cells were maintained on 1% gelatin-coated dishes in trations of cytokines, such as FGF2, did not stimulate to generate DMEM supplemented with 15% FCS͞10 mM nonessential amino spheres (Fig. 4B). However, we also observed that SU5402 (a acids͞0.1 mM 2-mercaptoethanol͞1,000 units/ml leukemia inhibi- pharmacological inhibitor of FGF receptor tyrosine kinases) and tory factor. anti-FGF2 antibodies blocked ES-derived neurosphere formation by cystatin C (see Fig. 10, which is published as supporting Primary Neurosphere Culture. The striatums of embryonic information on the PNAS web site). These results suggest that C57BL͞6 mice at day 12.5–14.0 were mechanically dissociated neurosphere formation by ES cells is stimulated by the cooperation and were cultured as suspension in DMEM͞F12 supplemented between endogenous FGFs and cystatin C. The importance of with 5 mM Hepes buffer͞0.3% glucose͞0.025 mg/ml insulin͞ endogenous FGF2 produced from ES cells for neural lineage 0.1 mg/ml transferrin͞20 nM progesterone͞0.06 mM pu- differentiation is in agreement with the study of Tropepe et al. (9). trescine͞30 nM sodium selenium (‘‘neurosphere medium’’) in Taken together, this hypothesis speculates that cystatin C secreted the presence of 20 ng/ml epidermal growth factor (EGF) from NSCs cooperates with endogenous FGFs, which have an (Sigma)͞10 ng/ml fibroblast growth factor 2 (FGF2) (Sigma) active role in the promotion of early neural differentiation of ES for 7 days (31). The concentration was set at 2.0–5.0 ϫ 105 cells cells. Taupin et al. (28) reported that cystatin C can be N- per ml. glycosylated and that this complex is required for its activity to induce FGF2-dependent proliferation of rat neuronal progenitor Coculture System. ES cells were washed three times and cocultured cells (NPCs) in vitro. Muotri et al. reported that L1 transcripts were in suspension with dissociated primary neurosphere cells derived enriched in N-glycosylated form of cystatin C-responsive NPCs, and from the striatums of embryonic mice at day 12.5–14.0 in neuro- L1 could retrotranspose during early neuronal differentiation, sphere medium. These mixed populations were cultured for 21 days affecting the expression of neuronal genes in vitro (29). This report in the presence of EGF (20 ng͞ml) and FGF2 (10 ng͞ml) individ- opens the possibility that N-glycosylation of cystatin C is required ually, in combination with, or in the absence of any exogenous for its ability to support differentiation of ES cells into NSCs. growth factors. Total concentration was set at 1.0 ϫ 106 cells per ml. However, the mechanism by which endogenous and exogenous During coculture, cells were dissociated and resuspended at day 7 FGFs effect the differentiation of ES cells into NSCs remains to be and 14. The number of round spheres or dissociated cells, which elucidated. were discriminated by GFP expression, were counted under the We demonstrated that cystatin C-treated ES-NSCs differen- microscope and spheres were defined as that measured Ͼ100 ␮m tiate into all four neuronal subtypes, including dopaminergic, in diameter. We defined ‘‘small cell clusters’’ as cell aggregations serotonergic, adrenergic, and cholinergic neurons (Fig. 4 E–H). measured Ͻ100 ␮m in diameter. Recently, there have been several reports on methods to produce For differentiation, single spheres were transferred to wells predominantly TH-positive neurons. Stromal cell-derived induc- coated with L-ornithine in 48-well culture plates and cultured in the ing activity produced TH-positive neurons at an efficiency of neurosphere medium containing 1% FCS in the absence of any 30% of Tuj-positive neurons (12), which is comparable to our growth factors. methods. In the current system, the activity of cystatin C was compared with several previously suspected neural-induction Cell Sorting. Fluorescence-activated cell sorter (FACS) sorting of factors (see Fig. 11, which is published as supporting information GFP-positive cells was performed on a FACS vantage flow cytom- on the PNAS web site). The results obtained suggested that eter͞cell sorter (Becton Dickinson). Cells (1–2 ϫ 106 per ml) were

cystatin C is superior to previously reported factors in generating analyzed for forward scatter, side scatter, propidium iodide (PI) NEUROSCIENCE neurospheres from ES cells. One of the notable features of our fluorescence, and GFP fluorescence with an argon laser (488 nm, system is the continuous expansion of cystatin C-treated ES- 100 mW). Dead cells were excluded by gating on forward and side NSCs that maintain a high differentiation activity during con- scatter and eliminating PI-positive events. D3 WT clones were used tinuous culture, making this system superior to previously re- to set the background fluorescence. Viable and fluorescent cells ported systems (11, 12, 30). Our system, cystatin C-treated were sorted into DMEM͞F12 medium at a speed of 1,000 cells per ES-NSCs, prove a good source for cell transplantation therapy, second. because cell replacement therapy needs large numbers of graft cells. In preliminary transplantation experiments, we demon- ES Cells Suspension Culture System. ES cells were three times washed strated that graft cells settled in and near the subventricular zone and cultured for 21 days at 1.0 ϫ 106 cells per ml in the presence

Kato et al. PNAS ͉ April 11, 2006 ͉ vol. 103 ͉ no. 15 ͉ 6023 Downloaded by guest on September 26, 2021 or absence of EGF (20 ng͞ml), FGF2 (10 ng͞ml) and CMPNC. the equivalents 2–4 ϫ 105 cells per ␮l. By using a blunt-ended 26G During culture, cells were dissociated and resuspended at days 7 Hamilton syringe, 1 ␮l of suspension medium was injected into the and 14. hemiventricular area of neonate mice. Self-renewal of ES-cell NSCs was determined by single-cell suspension culture as described in ref. 32. Dissociated ES cell- Protein Purification. The CMPNC (2 liters) was generated (see derived neurosphere cells were harvested and pelleted. The super- Supporting Methods, which is published as supporting informa- natant was aspirated, and the cell pellet was resuspended in tion on the PNAS web site). After concentrating 200-fold by neurosphere medium containing 0.3% agar with a cell density of ultrafiltration, CMPNC was semipurified through a phenyl hy- 200,000 cells per ml in the presence of FGF2 (10 ng͞ml) and EGF drophobic column (HiTrap, Amersham Pharmacia Biotech) by (20 ng͞ml) for 12 days. The number of round spheres was counted step-gradient elution with decreasing ammonium sulfate from 1 under the microscope at day 12. M to 0 M. For activity determination, each fractions eluted at the various concentration of ammonium sulfate were filtered (0.22 Immunocytochemistry and Immnunohistochemistry. Immunocyto- ␮m) and added in the medium of ES cells suspension culture chemistory was carried out by using standard protocols. Primary system. antibodies are listed in Table 2, which is published as supporting information on PNAS web site. The individual specificities of these Molecular Mass Spectrometric Analyses. Mass spectrometric iden- primary antibodies were tested by using appropriate tissues or cells tification of proteins was performed as described in ref. 33. as controls. Briefly, after SDS͞PAGE, proteins were visualized by silver For immunocytochemical staining of the sphere and brain tissues staining and excised separately from the gels, followed by the in serial section, they were fixed for 20 min at room temperature in in-gel digestions with trypsin (Promega) in a buffer containing 4% paraformaldehyde. After three rinses with PBS, the spheres 50 mM ammonium bicarbonate (pH 8.0) and 2% acetonitrile were equilibrated with 20% and 30% sucrose and sectioned (5–10 overnight at 37°C. Mass analyses of tryptic peptides were ␮ m) on a cryostat. Sections were incubated overnight at 4°C with performed by matrix-assisted laser desorption͞ionization primary antibodies. After rinsing three times with PBS, sections time-of-flight mass spectrometry (MALDI-TOF͞MS) with a were incubated for1hatroom temperature with secondary Voyager-DE͞STR (Applied Biosystems). Proteins were iden- antibodies. Appropriate cysnin-3-labeled (Jackson Immuno- tified by comparison between the molecular masses deter- Research) and Alexa-488-labeled (Molecular Probes) secondary mined by MALDI-TOF͞MS and theoretical peptide masses antibodies were used for visualization. from the proteins registered in NCBInr.

RNA Extraction and RT-PCR Analysis. Total cellular RNA was isolated HPLC Analysis. Dopamine release was measured by reverse phase by using the RNAeasy total RNA purification kit (Qiagen) followed HPLC, as described in ref. 34. Briefly, samples were collected at the by treatment with RNase-free RQ DNase. For cDNA synthesis, ͞ day of differentiation-conditioned medium (24 h), basal release (15 random hexamer primers (GIBCO BRL) were used to prime min in HBSS) and evoked release (15 min in HBSS ϩ 56 mM KCl). reverse transcriptase reactions. Using this method, it was possible Samples were stabilized and extracted by aluminum adsorption to use the same reverse transcriptase reaction (cDNA) for PCR (Chromosystem). Separation of injected samples (Autosampler amplification with different sets of gene-specific primers. The 540; ESA, Bedford, MA) was achieved by isocratic elution in the cDNA synthesis was carried out by using M-MLV SuperReverse MD-TM mobile phase (ESA) at 0.5 ml͞min. The oxidative poten- transcriptase (SuperScript II) at 42°C for 1 h. Primer sequences tial of the analytical cell (ESA Model 5011; Coulochem II, Bedford, (forward and reverse) and the length of the amplified products are MA) was set at 350 mV. Results were validated by coelution with listed in Table 2. Amplified products were electrophoresed in 2% catecholamine standards under varying buffer conditions and de- agarose gels containing ethidium bromide (25 ␮g͞ml), and band tector setting. were visualized with UV light. At least three replicates were performed. We thank Drs. S. Masuda and K. Inui for advice on HPLC analysis and Drs. T. Yasumi and H. Hiramatsu for critical reading of the manuscript. Transplantation Procedure. ES cells were cultured for 21 days in This work was supported by grants from the Science Reseach on Priority chemically defined medium by adding the CMPNC in the presence Areas; the Creative Science Research; the Japan Society for the Pro- of FGF2 and EGF. At the end of culture, ES-derived spheres were motion of Science; and the Ministry of Education, Culture, Sports, dissociated, washed twice, and then suspended in DMEM͞F12 at Science, and Technology.

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