Statistical Analysis All quantitative measurements were collected with N=3�C4 independent determinations per data point and expressed as mean �� standard deviation. Data for these measurements were analyzed with Microsoft Excel software utilizing a Student’s two-tailed t-test assuming equal levels thoroughly of variance. Statistically significant values were defined as p<0.05. Ethics Statement All animals were handled in strict accordance with good animal practice as defined by the standards outlined in the National Research Council's Guide and Children's Hospital Boston (CHB) PHS Assurance, and all animal work was approved by the CHB Animal Care and Use Committee (IACUC, Protocol 08-09-1207). Results Effect of RA on ESC UP Expression RA is known to function in both a time- and concentration-dependent manner to selectively regulate lineage specification during ESC differentiation [27], [40], [41].

We first investigated the ability of continuous cultivation of ESCs in the presence of RA (0.01�C10 ��M) to stimulate urothelial marker expression. Real time RT-PCR analysis demonstrated that withdrawal of LIF as well as the addition of micromolar concentrations of RA led to the down-regulation of OCT-4 mRNA transcript levels in ESC cultures, Cilengitide indicating a progression toward differentiation (Figure 1A). In addition, RA acted synergistically with LIF withdrawal to promote pluripotency marker decline. RA stimulation increased mRNA transcript levels of all five uroplakins (UPs) over those seen in spontaneously differentiating controls and na?ve ESCs by day 6 of cultivation with significant elevation occurring by 9 d and continuing to rise through 14 d of culture (Figure S1). The ability of RA to stimulate UP expression was concentration-dependent, with micromolar concentrations stimulating maximal extents of mRNA transcription over the 9 d time course (Figure 1B). Figure 1 RA stimulation of ESCs induces UP expression in a time and concentration-dependent manner.