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

Vaajasaari H, Ilmarinen T, Juuti-Uusitalo K, Rajala K, Onnela N, Narkilahti S, Suuronen R, Hyttinen J, Uusitalo H, Skottman H - Mol. Vis. (2011)

Bottom Line: The expression of RPE-specific markers was confirmed at the gene and protein level.Moreover, we introduced an improved method to generate functional putative RPE cells without xeno-components under defined conditions.Our results demonstrate that putative hESC-RPE and hiPSC-RPE express genes and proteins characteristic for RPE cells, as well as being able to phagocytose POS and secrete PEDF.

View Article: PubMed Central - PubMed

Affiliation: Regea-Institute for Regenerative Medicine, University of Tampere, Tampere, Finland.

ABSTRACT

Purpose: The production of functional retinal pigment epithelium (RPE) cells from human embryonic (hESCs) and human induced pluripotent stem cells (hiPSCs) in defined and xeno-free conditions is highly desirable, especially for their use in cell therapy for retinal diseases. In addition, differentiated RPE cells provide an individualized disease model and drug discovery tool. In this study, we report the differentiation of functional RPE-like cells from several hESC lines and one hiPSC line in culture conditions, enabling easy translation to clinical quality cell production under Good Manufacturing Practice regulations.

Methods: Pluripotent stem cells were cultured on human fibroblast feeder cells in serum-free medium. The differentiation toward RPE was induced by removing basic fibroblast growth factor and feeder cells from the serum-free conditions. RPE differentiation was also achieved using xeno-free and defined culture conditions. The RPE cell morphology and pigmentation of the cells were analyzed and the expression of genes and proteins characteristic for RPE cells was evaluated. In vitro functionality of the cells was analyzed using ELISA measurements for pigment epithelium derived factor (PEDF) secretion and phagocytosis of photoreceptor outer segments (POS). The integrity of the generated RPE layers was analyzed using transepithelial electric resistance measurements.

Results: We generated putative RPE cells with typical pigmented cobblestone-like morphology. The expression of RPE-specific markers was confirmed at the gene and protein level. The differentiated cells were able to phagocytose POS and secrete PEDF characteristic of native RPE cells. In addition, cultured cells formed a polarized epithelium with high integrity and exhibited excellent transepithelial electric resistance values, indicating well established, tight junctions. Moreover, we introduced an improved method to generate functional putative RPE cells without xeno-components under defined conditions.

Conclusions: We have developed a progressive differentiation protocol for the production of functional RPE-like cells from hESCs and hiPSCs. Our results demonstrate that putative hESC-RPE and hiPSC-RPE express genes and proteins characteristic for RPE cells, as well as being able to phagocytose POS and secrete PEDF. Furthermore, our results show that RPE-like cells can be differentiated in xeno-free and defined culture conditions, which is mandatory for Good Manufacturing Practice-production of these cells for clinical use.

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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.
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f7: 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.

Mentions: RT–PCR and immunostainings were performed for the cells (Regea 08/023 and FiPS 5–7) differentiated in RPEregES medium. On D44, cells from both cell lines expressed all analyzed eye/RPE-specific markers (PAX6, RAX, MITF, RPE65, bestrophin, OTX2v1, PMEL, PEDF, tyrosinase; Regea 08/023, Figure 7A; FiPS 5–7, data not shown). The pigmented cells in RPEregES conditions were manually selected on D51. Their morphology and protein expression were analyzed on D83. The pigmented cells differentiated from both hESC and hiPSC lines had cobblestone-like morphology, which is typical for RPE cells. In addition, the cells were positive for RPE65, ZO-1, MITF, CRALBP, Bestrophin, and Na+/K+ATPase, which are important for the functionality of the RPE cell. There were some Ki67 positive cells, which were clearly more immature according to the cell morphology (Regea 08/023, Figure 7B-I; FiPS 5–7, data not shown). In addition to gene and protein expression, putative RPE cells differentiated with RPEregES secreted PEDF. On average, PEDF secretion after 49 days of differentiation was 16.2 ng/ml and 97 days was 182.4 ng/ml from hESC-RPE (Regea 08/023) cells. Corresponding values from hiPSC-RPE (FiPS 5–7) differentiated with the RPEregES method were 10.6 ng/ml and 463.15 ng/ml.


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

Vaajasaari H, Ilmarinen T, Juuti-Uusitalo K, Rajala K, Onnela N, Narkilahti S, Suuronen R, Hyttinen J, Uusitalo H, Skottman H - Mol. Vis. (2011)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3044694&req=5

f7: 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.
Mentions: RT–PCR and immunostainings were performed for the cells (Regea 08/023 and FiPS 5–7) differentiated in RPEregES medium. On D44, cells from both cell lines expressed all analyzed eye/RPE-specific markers (PAX6, RAX, MITF, RPE65, bestrophin, OTX2v1, PMEL, PEDF, tyrosinase; Regea 08/023, Figure 7A; FiPS 5–7, data not shown). The pigmented cells in RPEregES conditions were manually selected on D51. Their morphology and protein expression were analyzed on D83. The pigmented cells differentiated from both hESC and hiPSC lines had cobblestone-like morphology, which is typical for RPE cells. In addition, the cells were positive for RPE65, ZO-1, MITF, CRALBP, Bestrophin, and Na+/K+ATPase, which are important for the functionality of the RPE cell. There were some Ki67 positive cells, which were clearly more immature according to the cell morphology (Regea 08/023, Figure 7B-I; FiPS 5–7, data not shown). In addition to gene and protein expression, putative RPE cells differentiated with RPEregES secreted PEDF. On average, PEDF secretion after 49 days of differentiation was 16.2 ng/ml and 97 days was 182.4 ng/ml from hESC-RPE (Regea 08/023) cells. Corresponding values from hiPSC-RPE (FiPS 5–7) differentiated with the RPEregES method were 10.6 ng/ml and 463.15 ng/ml.

Bottom Line: The expression of RPE-specific markers was confirmed at the gene and protein level.Moreover, we introduced an improved method to generate functional putative RPE cells without xeno-components under defined conditions.Our results demonstrate that putative hESC-RPE and hiPSC-RPE express genes and proteins characteristic for RPE cells, as well as being able to phagocytose POS and secrete PEDF.

View Article: PubMed Central - PubMed

Affiliation: Regea-Institute for Regenerative Medicine, University of Tampere, Tampere, Finland.

ABSTRACT

Purpose: The production of functional retinal pigment epithelium (RPE) cells from human embryonic (hESCs) and human induced pluripotent stem cells (hiPSCs) in defined and xeno-free conditions is highly desirable, especially for their use in cell therapy for retinal diseases. In addition, differentiated RPE cells provide an individualized disease model and drug discovery tool. In this study, we report the differentiation of functional RPE-like cells from several hESC lines and one hiPSC line in culture conditions, enabling easy translation to clinical quality cell production under Good Manufacturing Practice regulations.

Methods: Pluripotent stem cells were cultured on human fibroblast feeder cells in serum-free medium. The differentiation toward RPE was induced by removing basic fibroblast growth factor and feeder cells from the serum-free conditions. RPE differentiation was also achieved using xeno-free and defined culture conditions. The RPE cell morphology and pigmentation of the cells were analyzed and the expression of genes and proteins characteristic for RPE cells was evaluated. In vitro functionality of the cells was analyzed using ELISA measurements for pigment epithelium derived factor (PEDF) secretion and phagocytosis of photoreceptor outer segments (POS). The integrity of the generated RPE layers was analyzed using transepithelial electric resistance measurements.

Results: We generated putative RPE cells with typical pigmented cobblestone-like morphology. The expression of RPE-specific markers was confirmed at the gene and protein level. The differentiated cells were able to phagocytose POS and secrete PEDF characteristic of native RPE cells. In addition, cultured cells formed a polarized epithelium with high integrity and exhibited excellent transepithelial electric resistance values, indicating well established, tight junctions. Moreover, we introduced an improved method to generate functional putative RPE cells without xeno-components under defined conditions.

Conclusions: We have developed a progressive differentiation protocol for the production of functional RPE-like cells from hESCs and hiPSCs. Our results demonstrate that putative hESC-RPE and hiPSC-RPE express genes and proteins characteristic for RPE cells, as well as being able to phagocytose POS and secrete PEDF. Furthermore, our results show that RPE-like cells can be differentiated in xeno-free and defined culture conditions, which is mandatory for Good Manufacturing Practice-production of these cells for clinical use.

Show MeSH
Related in: MedlinePlus