Additional file 2: Figure S2. of A strategy to ensure safety of stem cell-derived retinal pigment epithelium cells

Showing expression of indicated proteins measured by flow cytometry in hESCs (left) and hESC-derived RPE (right). x axis represents the log-fluorescence intensity, y axis represents relative cell counts. (TIF 1923 kb

Additional file 1: Figure S1. of A strategy to ensure safety of stem cell-derived retinal pigment epithelium cells

Showing a representative bright-field image of typical morphology of cells used for screening for cell surface markers. The cells form a monolayer and display cobblestone morphology typical of RPE cells. Scale bar = 400 μm (10×) and 200 μm (20×). (TIF 8794 kb

Additional file 4: Figure S4. of A strategy to ensure safety of stem cell-derived retinal pigment epithelium cells

Showing expression of CD59 (top) and TRA-1-60 (bottom) measured by flow cytometry in cell suspensions created by mixing together different hESC-derived RPE and hESCs. x axis represents the log-fluorescence intensity, y axis represents relative cell counts. (TIF 2661 kb

Additional file 5: Figure S5. of A strategy to ensure safety of stem cell-derived retinal pigment epithelium cells

Showing the CD59+ and CD59– samples sorted in Fig. 3 re-analysed by flow cytometry to show the purity of the individual fractions collected. The P4 gated events represent the CD59-positive fraction and the P5 gated events represent the CD59-negative fraction. (TIF 2445 kb

Additional file 3: Figure S3. of A strategy to ensure safety of stem cell-derived retinal pigment epithelium cells

Showing expression of CD59 measured by flow cytometry at days 0, 6, 9 and 45 of the differentiation time course. x axis represents the log-fluorescence intensity, y axis represents relative cell counts. (TIF 1704 kb

A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

<div><p>The integrity of the epithelium is maintained by a complex but regulated interplay of processes that allow conversion of a proliferative state into a stably differentiated state. In this study, using human embryonic stem cell (hESC) derived Retinal Pigment Epithelium (RPE) cells as a model; we have investigated the molecular mechanisms that affect attainment of the epithelial phenotype. We demonstrate that RPE undergo a Mesenchymal–Epithelial Transition in culture before acquiring an epithelial phenotype in a FOXM1 dependent manner. We show that FOXM1 directly regulates proliferation of RPE through transcriptional control of cell cycle associated genes. Additionally, FOXM1 modulates expression of the signaling ligands BMP7 and Wnt5B which act reciprocally to enable epithelialization. This data uncovers a novel effect of FOXM1 dependent activities in contributing towards epithelial fate acquisition and furthers our understanding of the molecular regulators of a cell type that is currently being evaluated as a cell therapy.</p></div

Epithelial fate acquisition is density dependent.

<p>A. Quantification of change in cell density (number of DAPI positive nuclei per cm² imaged area) upon FOXM1 overexpression or knockdown, 72h post transfection. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 4). P<0.0001 (Student’s t-test). B. Heatmap showing changes in gene expression of a panel of representative markers over a timecourse of RPE culture where cells are seeded at high (100000 cells/cm²) or low (8000 cells/cm²) density. C. Plot showing differential expression of <i>BMP7</i> and <i>Wnt5B</i> transcripts extrapolated from the microarray data. The shaded area represents 95% confidence intervals around the point estimates (circles) of the difference between the mean high density expression vs the mean low density expression.</p

FOXM1 promotes RPE epithelial fate.

<p>A. qPCR based measurement of transcript expression of a panel of epithelial (red) and mesenchymal (green) markers at Day 10 post siFOXM1 transfection (except levels of <i>FOXM1</i> itself which are measured at Day 2 post knockdown). Data is normalized to transfection with non-targeting siRNA used as a control. <i>ACTB</i>, <i>GAPDH</i>, <i>IPO8</i> and <i>HPRT1</i> are used as housekeeping genes. Bars represent Mean + SD (n = 3). P<0.05 (Student’s t-test). B. Immunocytochemistry for PMEL17 upon FOXM1 knockdown (siFOXM1) or overexpression (pFOXM1) at Day 10 post transfection. C. Level of knockdown obtained upon transient transfection of siRNA against SNAI2, SNAI1, ZEB1, TWIST1 and GSC. Knockdown was measured by qPCR at Day 6 post transfection and is expressed relative to non-targeting siRNA used as control. <i>CYC1</i> and <i>GAPDH</i> were used as housekeeping genes. Bars represent Mean ± SD (n = 6–9). Knockdown of EMT-TF expression was significant, P<0.05 (Student’s t-test). D. No significant effect on <i>PMEL</i>, <i>MITF</i> or <i>BEST1</i> expression was observed under the same conditions described above for Fig 2C.</p

Model showing proposed roles of FOXM1 in epithelial fate acquisition.

<p>RPE first acquire a mesenchymal morphology upon dissociation and culture followed by proliferation and mesenchymal-epithelial transition to re-uptake an epithelial phenotype. Proliferation of RPE is directly regulated by FOXM1 which also affects expression of BMP7 and Wnt5B by an unknown mechanism. Both these activities are required for successful MET and epithelialization.</p

FOXM1 regulates RPE proliferation.

<p>A. Graph showing quantification of immunocytochemistry where % Ki67 (n = 3) or % EdU (n = 6) is plotted on the left Y axis and relative expression of <i>FOXM1</i> transcript (n = 3; <i>ACTB</i> used as housekeeping gene) on the right Y axis over days in culture (x axis). B. Quantification of change in <i>FOXM1</i> transcript upon transient overexpression (pFOXM1) or knockdown (siFOXM1), 48h post transfection, measured by qPCR. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 3). C. Quantification of change in EdU incorporation upon FOXM1 overexpression or knockdown, 72h post transfection. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 4). P<0.0001 (Student’s t-test). D. Quantification of immunocytochemistry for Ki67 upon siRNA mediated knockdown of non-targeting control, GAPDH, SNAI1, SNAI2 and FOXM1, at Day 6 post transfection. Bars represent Mean + SD (n = 3). n.s non-significant, * p<0.05 Student’s t-test. E. Effect of Thiostrepton on EdU incorporation [left Y axis, red] and <i>FOXM1</i> transcript expression measured by qPCR [right Y axis, blue]. Bars represent Mean ± SD (n = 6). F. Bright-field microscopy showing a scratch introduced in a RPE monolayer at 0 hrs and 19hrs in the presence of DMSO or 10μM Thiostrepton. Edge of the scratch is marked with a white line. Scale bar = 200 μm. G. Quantification of F (above). Bars represent Mean + SD (n = 7). P<0.0001 (Student’s t-test)</p