title: Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells.
abstract: OBJECTIVE
  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.
authors.affiliation: Regea-Institute for Regenerative Medicine, University of Tampere, Tampere, Finland.
authors.forename: Hanna
authors.initials: H
authors.lastname: Vaajasaari
authors.forename: Tanja
authors.initials: T
authors.lastname: Ilmarinen
authors.forename: Kati
authors.initials: K
authors.lastname: Juuti-Uusitalo
authors.forename: Kristiina
authors.initials: K
authors.lastname: Rajala
authors.forename: Niina
authors.initials: N
authors.lastname: Onnela
authors.forename: Susanna
authors.initials: S
authors.lastname: Narkilahti
authors.forename: Riitta
authors.initials: R
authors.lastname: Suuronen
authors.forename: Jari
authors.initials: J
authors.lastname: Hyttinen
authors.forename: Hannu
authors.initials: H
authors.lastname: Uusitalo
authors.forename: Heli
authors.initials: H
authors.lastname: Skottman
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D048430:Cell Shape; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D009928:Organ Specificity / Q000235:genetics; D010587:Phagocytosis; D010858:Pigmentation / Q000502:physiology; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology*
chemical_list: D005136:Eye Proteins