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

Abstract

Although embryonic stem (ES) cells are capable of unlimited proliferation and pluripotent differentiation, effective preparation of neural stem cells from ES cells are not achieved. Here, we have directly generated under the coculture with dissociated primary neurosphere cells in serum-free medium and the same effect was observed when ES cells were cultured with conditioned medium of primary neurosphere culture (CMPNC). ES-neural stem cells (NSCs) could proliferate for more than seven times and differentiate into neurons, astrocytes, and oligodendrocytes in vitro and in vivo. The responsible molecule in CMPNC was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, which turned out to be cystatin C. Purified cystatin C in place of the CMPNC could generate ES-NSCs efficiently with self-renewal and multidifferentiation potentials. These results reveal the validity of cystatin C for generating NSCs from ES cells.

Fig. 1.

ES cells differentiate into NSCs by coculture with dissociated primary neurosphere cells. (A) ES cells, which constitutively express GFP, cocultured with dissociated primary neurosphere cells form spheres in the presence of FGF2 (10 ng/ml) and EGF (20 ng/ml) after 21 days (Scale bars: 100 μm.) (B) ES cells could generate spheres efficiently (filled bar). However, when ES cells were cultured alone, the majority of ES cells died and only a few small cell clusters were generated (open bar). Data are means ± SD of triplicate determinations from two or three independent experiments. (C–H) Differentiated ES-derived spheres contain neurons (C, MAP2), astrocytes (D, GFAP), and oligodendrocytes (E, galactocerebroside). ES-derived spheres passaged five times retained also multilineage potential (F, Tuj; G, MAP2; H, MBP). (Scale bar: 50 μm.) (I) ES-derived spheres could be served to the repeated propagation. (J) The expression of Nestin, Tuj, GFAP, and Gal C genes in primary, twice-passaged, and four times-passaged ES-derived spheres was determined by RT-PCR. Lanes: 1, negative control; 2, primary ES-derived spheres; 3, P2, twice-passaged ES-derived spheres; 4, P4, four times-passaged ES-derived spheres.

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 ± SD of triplicate determinations from two or three independent experiments.

Fig. 3.

CMPNC-treated ES-derived spheres had multilineage differentiation potential and self-renewal potential. Differentiated ES-derived spheres contain nestin-positive cells (A, Cy3-nestin). ES-derived spheres could differentiate into neurons (B and D, Cy3-Tuj; C, MAP2), astrocytes (D and E, FITC-GFAP), oligodendrocytes (E, Cy3-MBP; F, Cy3-Gal C). They could differentiated into various matured neurons (G, Cy3-TH; H, Cy3-DAT/FITC-Tuj; I, Cy3-ChAT; J, Cys-3-DBH; K, Cy3-Serotonin). Transplantation of ES cell-derived neurospheres into the neonatal mouse brain. All grafts were easily detected by the GFP expression as ES-derived cells (M) as compared with control (L). In serial sections, TH-positive cells (N, Cy3-TH) and GFAP-positive cells (O, Cy3-GFAP) with GFP expression could be detected around the subventricular zone. (P) A time course study of the appearance of ES-derived neurospheres with Tuj, TH, DBH, and serotonin-like immunoreactivities. Positive sphere was defined as a sphere containing 10 or more cells with immunoreactivity of each antibody. (Q) ES-derived spheres could be served to the repeated propagation in the presence of FGF2 and EGF. (R) Single-cell suspension culture in serum-free medium containing 0.3% agar were prepared. Sequential images of a single CMPNC-treated ES-derived cell at 1.5, 4, and 12 days after culturing. (Scale bars: 50 μm.)

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-Tuj/FITC-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 μm.) (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 ± SD of triplicate determinations from two or three independent experiments.

Fig. 5.

Cystatin C supported to differentiate into NSCs from ES cells. (A and B) Immunocytochemical analysis of nestin-positive cells constituted small cell clusters and spheres, which developed either in the absence or presence of cystatin C. (C and D) Significantly more Tuj-positive cells differentiated from spheres developed in the presence of cystatin C than small cell clusters developed in the absence of cystatin C. (Scale bars: 100 μm.) (E) Immunocytochemical analysis of Tuj-, GFAP-, and MBP-positive cells differentiated from spheres developed in the presence of cystatin C and small cell clusters developed in the absence of cystatin C at day 3 and day 7. Data are means ± SD of triplicate determinations from two or three independent experiments. (F) Comparisons were made about the expression of Rex1, nestin, Nurr1, and En1 in the cystatin C-treated and cystatin C-untreated ES-derived sphere by RT-PCR. Lanes: 1, negative control; 2, ES cells; 3, primary neurospheres; 4, cystatin C-treated ES cell-derived spheres; 5, cystatin C untreated ES cell-derived small cell clusters.