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1.
Figure 4

Figure 4. From: Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells.

Retina and anterior neural fold homeobox (RAX) expression by FiPS 5–7 during differentiation. Undifferentiated cells (D0) have been used as a reference sample.

Hanna Vaajasaari, et al. Mol Vis. 2011;17:558-575.
2.
Figure 2

Figure 2. From: Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells.

A schematic illustration of implementation of the study. Analyses which were done from the RPEregES condition besides the RPEbasic condition are marked with asterisks (*). Analyses were done from floating aggregate cultures unless marked with gray boxes for adherent culture.

Hanna Vaajasaari, et al. Mol Vis. 2011;17:558-575.
3.
Figure 1

Figure 1. From: Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells.

Differentiation of human pluripotent stem cells toward retinal pigment epithelium cells. A: A schematic representation of retinal pigment epithelium (RPE) cell differentiation during retinal development. B: Reverse transcription (RT)–PCR analysis of typical genes for retinal development expressed during putative RPE differentiation of the human embryonic stem cell (hESC) line Regea 08/023 and human induced pluripotent stem cell (hiPSC) line FiPS 5–7 at sequential time points on D7–D72.

Hanna Vaajasaari, et al. Mol Vis. 2011;17:558-575.
4.
Figure 6

Figure 6. From: Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells.

Phagocytosis of photoreceptor outer segments (POS) and cell membrane polarization of human embryonic stem cell (hESC; Regea 08/023) and human induced pluripotent stem cell (hiPSC; FiPS 5–7)-derived retinal pigment epithelium (RPE) cells. A: Putative hESC-RPE and B: hiPSC-RPE internalize POS (green, arrowheads) for cell morphology; F-actins were stained using phalloidin (red). Vertical confocal sections showing apical localization of Na+/K+ATPase (green) and basolateral localization of Bestrophin (red) in C: hESC-RPE and D: hiPSC-RPE. Images were taken with an LSM 700 confocal microscope (Carl Zeiss) using a 63× oil immersion objective, scale bar 20 μm.

Hanna Vaajasaari, et al. Mol Vis. 2011;17:558-575.
5.
Figure 3

Figure 3. From: Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells.

Immunofluorescence staining of human embryonic stem cell (hESC; Regea 08/023)- and human induced pluripotent stem cell (hiPSC; FiPS 5–7)-derived retinal pigment epithelium cells revealing maturation stage after 83 days of differentiation. Cellular retinaldehyde-binding protein (CRALBP) and microphthalmia-associated transcription factor (MITF) localization in A-C: manually selected hESC-RPE cells and D-F: hiPSC-retinal pigment epithelium (RPE) cells. G, I: RPE65 expression in hESC-RPE and J, L: hiPSC-RPE. H, K: For cell morphology, F-actins were stained using phalloidin. Tight junction protein anti-zonula occludens (ZO)-1 and proliferation marker Ki67 localization in M: hESC-RPE cells and N: hiPSC-RPE cells. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Images A-F were taken with an Olympus BX60 microscope (Olympus, Tokyo, Japan) using a 60× oil immersion objective, scale bar 20 μm. Images G-N were taken with an LSM 700 confocal microscope (Carl Zeiss) using a 63× oil immersion objective, scale bar 20 μm.

Hanna Vaajasaari, et al. Mol Vis. 2011;17:558-575.
6.
Figure 7

Figure 7. From: Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells.

Differentiation of human pluripotent stem cells toward retinal pigment epithelium (RPE) cells under defined culture conditions, RPEregES. All represented images are from human embryonic stem cell (hESC)-RPE Regea 08/023. A: Reverse transcription (RT)–PCR analysis of typical genes for retinal/ RPE development expressed by undifferentiated hESC (Regea 08/023), human foreskin fibroblast (hFF) feeder cells, and putative hESC-RPE on D7 and D44. Expression of B: Microphthalmia-associated transcription factor (MITF), B: Cellular retinaldehyde-binding protein (CRALBP), and E,G: RPE65 on D83. F: For cell morphology, F-actins were stained using phalloidin. H: Proliferative activity was studied by Ki67 staining together with tight junction protein anti-zonula occludens (ZO)-1 in hESC-RPE. I: Vertical confocal sections showing apical localization of Na+/K+ATPase (green) and basolateral localization of Bestrophin (red). Nuclei stained with 4',6-diamidino-2-phenylindole (DAPI). Images B-D were taken with an Olympus BX60 microscope (Olympus, Tokyo, Japan) using a 60× oil immersion objective, scale bar 20 μm. Images E-I were taken with an LSM 700 confocal microscope (Carl Zeiss) using a 63× oil immersion objective, scale bar 20 μm.

Hanna Vaajasaari, et al. Mol Vis. 2011;17:558-575.
7.
Figure 5

Figure 5. From: Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells.

Morphology and gene expression analysis of manually selected and long-term cultured human embryonic stem cell (hESC)-retinal pigment epithelium (RPE; Regea 08/023) and hiPSC-RPE (FiPS 5–7) cells. A: Bright-field micrograph of hESC-retinal pigment epithelium (RPE) and human induced pluripotent stem cell (hiPSC)-RPE cells cultured for 136 days on Collagen IV. The cells have acquired a cobblestone morphology and a high degree of pigmentation, which is typical of RPE cells. Low magnification images were captured with a Nikon Eclipse TE2000-S phase contrast microscope (Nikon Instruments Europe B.V. Amstelveen, The Netherlands) and higher magnification images with an Olympus BX60 microscope (Olympus, Tokyo, Japan) using a 60× oil immersion objective. Scale bar 20 μm. B: Reverse transcription (RT)–PCR analysis showing the expression of optic vesicle, optic cup, RPE, neural crest melanocyte, pluripotent stem cell, mesoderm and endoderm marker genes by undifferentiated cells (D0), human foreskin fibroblast (hFF) feeder cells, and putative RPE cells (D196) from Regea 08/023 and FiPS 5–7 cells. N/A=not analyzed. C: Relative OCT3/4 expression between undifferentiated FiPS 5–7 and putative hiPSC-RPE after 196 days of differentiation.

Hanna Vaajasaari, et al. Mol Vis. 2011;17:558-575.

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