Figure 2

Msi1-ffLuc expression corresponds with Msi1-positive NS/PCs and astrocytes in Msi1-ffLuc ESC-derived neural cells. (A) The experimental protocol for embryonic stem cell (ESC) differentiation with a retinoic-acid (RA) conditioned medium. ESCs formed embryoid bodies (EB) after 6 days in floating culture. (B) The expression of Msi1-ffLuc on day 0 of ESCs and day 6 of EBs is shown in the left panels. GFP was not expressed in ESCs and RA-untreated EBs(-RA) but was expressed in RA-treated EB(+RA). Nine Msi1-ffLuc ES cell lines were analysed for GFP fluorescence in each condition indicated (right panel). Scale bar: 100 μm. FLU: firefly luciferase light unit. (C) Dissociated day-6 EBs (+RA) derived from Msi1-ffLuc ES cell line (Red line in left panel) were assessed by flow cytometry and divided into H, M, and L fractions according to their GFP intensity. Each fraction was then analysed by qRT-PCR. The GFP, Msi1, and Nestin mRNA transcripts were enriched in the H fraction (L = 1 in each fraction). Black line in left panel shows day-6 EBs (+RA) derived from wild type ES cell line. (D), (E) secondary neurospheres were dissociated, cultured in differentiation medium for 4 days, and immunostained. Anti-GFP reactivity for GFP(+) cells correlated with Nestin(+) and endogenous Msi1(+) cells, but not with MAP2(+) mature neurons with long processes (D, Scale bar: 50 um). Vertical arrowheads show Marker(+)/GFP(+) cells. The immunofluorescent intensity of each marker was represented by scatter blots (E). The vertical axis shows the intensity of Msi1 and of cell-type-specific markers for neurons (β-III Tubulin), astrocytes (GFAP) and NS/PCs (Nestin). The horizontal axis shows the endogenous Msi1 (upper panel) and GFP (lower panel) intensities. The proportion (%) of marker(+) cells among GFP(+) cells is indicated in the bottom of each box. Note that the GFP/Marker double-positive cells were highly observed for Msi1, GFAP, and Nestin, but the β-III Tubulin-positive neurons had weak GFP expression.