FIG. 4.
Differentiation of ES cells lacking LSD1 is associated with increased cell death and a perturbed transcriptional program. (A) Differentiation potential. ES cells of the indicated genotype were plated at low density in the presence or absence of LIF and cultured for 6 days before staining for the presence of alkaline phosphatase and scored as undifferentiated (intense purple), mixed (weak purple), or differentiated (no staining). (B) Colonies were stained with methylene blue and counted. Mean values are plotted (n = 3) ± SEM. (C) The left panel shows images representative of embryoid bodies (EBs) at 1 and 5 days of culture, and the right panel shows the number of EBs of the indicated genotype obtained after 5 days of culture. (D) The percentage of dead cells was determined by propidium iodide staining and FACS analysis, after treatment with 1 μM retinoic acid. The percentage of cells with a sub-G1 DNA content is indicated. (E) Quantitative RT-PCR data for genes characteristic of undifferentiated stem cells (Oct-4, Nanog, and Rex1), endoderm (Gata6), primitive ectoderm (Fgf5), and mesoderm (brachyury), was performed as indicated on mRNA collected at days 0, 2, and 5 during EB differentiation. Mean values (n = 3) ± SEM are plotted. Values indicate expression of the specific gene relative to GAPDH measured using Universal ProbeLibrary hydrolysis probes. (F) Representative bright-field image (top panel) and average size of EBs (bottom panel) after 12 days of culture. Arrows indicate clumps of dead cells which accumulate in Lsd1Δ3/Δ3 cultures. Scale bar = 300 μm. The bottom panel shows the diameter of EBs of the indicated genotype obtained after 12 days of culture. (G) Quantitative RT-PCR was performed as in panel E for markers of endoderm (TTR, AFP, and Foxa2), adipose (FABP4), and cardiomyocyte (Mef2c) tissue. mRNA was isolated from EBs of the indicated genotype at 0, 5, 12, and 15 days of culture.