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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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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.
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f6: 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.

Mentions: The functionality of putative hESC-RPE (Regea 08/023) and hiPSC-RPE (FiPS 5–7) was shown by POS phagocytosis, PEDF secretion, polarization of cells, and the integrity of the epithelial structure by TEER measurements. The results show that pigmented cells from both lines were able to phagocytose POS (Figure 6A,B), as opposed to nonpigmented cells, which were used as controls (data not shown).


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)

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

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

f6: 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.
Mentions: The functionality of putative hESC-RPE (Regea 08/023) and hiPSC-RPE (FiPS 5–7) was shown by POS phagocytosis, PEDF secretion, polarization of cells, and the integrity of the epithelial structure by TEER measurements. The results show that pigmented cells from both lines were able to phagocytose POS (Figure 6A,B), as opposed to nonpigmented cells, which were used as controls (data not shown).

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