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 neurons, astrocytes, and oligodendrocytes 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 central nervous system (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- astrocyte marker glial fibrillary acidic protein (GFAP) (Fig. 1D) stricted. It has been shown that neural fates emerge from ES cells and oligodendrocyte 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 neuron 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 stem cell; 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, neural stem cell; 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: