PURPOSE: To determine the effects of serial expansion on the cellular, molecular, and functional properties of human iPS cell (hiPSC)-derived RPE cultures. METHODS: Fibroblasts obtained from four individuals were reprogrammed into hiPSCs and differentiated to RPE cells using previously described methods. Patches of deeply pigmented hiPSC-RPE were dissected, dissociated, and grown in culture until they re-formed pigmented monolayers. Subsequent passages were obtained by repeated dissociation, expansion, and maturation of RPE into pigmented monolayers. Gene and protein expression profiles and morphological and functional characteristics of hiPSC-RPE at different passages were compared with each other and to human fetal RPE (hfRPE). RESULTS: RPE from all four hiPSC lines could be expanded more than 1000-fold when serially passaged as pigmented monolayer cultures. Importantly, expansion of hiPSC-RPE monolayers over the first three passages (P1-P3) resulted in decreased expression of pluripotency and neuroretinal markers and maintenance of characteristic morphological features and gene and protein expression profiles. Furthermore, P1 to P3 hiPSC-RPE monolayers reliably demonstrated functional tight junctions, G-protein-coupled receptor-mediated calcium transients, phagocytosis and degradation of photoreceptor outer segments, and polarized secretion of biomolecules. In contrast, P4 hiPSC-RPE cells failed to form monolayers and possessed altered morphological and functional characteristics and gene expression levels. CONCLUSIONS: Highly differentiated, pigmented hiPSC-RPE monolayers can undergo limited serial expansion while retaining key cytological and functional attributes. However, passaging hiPSC-RPE cultures beyond senescence leads to loss of such features. Our findings support limited, controlled passaging of patient-specific hiPSC-RPE to procure cells needed for in vitro disease modeling, drug screening, and cellular transplantation.
PURPOSE: To determine the effects of serial expansion on the cellular, molecular, and functional properties of human iPS cell (hiPSC)-derived RPE cultures. METHODS: Fibroblasts obtained from four individuals were reprogrammed into hiPSCs and differentiated to RPE cells using previously described methods. Patches of deeply pigmented hiPSC-RPE were dissected, dissociated, and grown in culture until they re-formed pigmented monolayers. Subsequent passages were obtained by repeated dissociation, expansion, and maturation of RPE into pigmented monolayers. Gene and protein expression profiles and morphological and functional characteristics of hiPSC-RPE at different passages were compared with each other and to human fetal RPE (hfRPE). RESULTS: RPE from all four hiPSC lines could be expanded more than 1000-fold when serially passaged as pigmented monolayer cultures. Importantly, expansion of hiPSC-RPE monolayers over the first three passages (P1-P3) resulted in decreased expression of pluripotency and neuroretinal markers and maintenance of characteristic morphological features and gene and protein expression profiles. Furthermore, P1 to P3 hiPSC-RPE monolayers reliably demonstrated functional tight junctions, G-protein-coupled receptor-mediated calcium transients, phagocytosis and degradation of photoreceptor outer segments, and polarized secretion of biomolecules. In contrast, P4 hiPSC-RPE cells failed to form monolayers and possessed altered morphological and functional characteristics and gene expression levels. CONCLUSIONS: Highly differentiated, pigmented hiPSC-RPE monolayers can undergo limited serial expansion while retaining key cytological and functional attributes. However, passaging hiPSC-RPE cultures beyond senescence leads to loss of such features. Our findings support limited, controlled passaging of patient-specific hiPSC-RPE to procure cells needed for in vitro disease modeling, drug screening, and cellular transplantation.
Authors: Hans E Grossniklaus; Jun X Ling; Timothy M Wallace; Stefan Dithmar; Diane H Lawson; Cynthia Cohen; Victor M Elner; Susan G Elner; Paul Sternberg Journal: Mol Vis Date: 2002-04-21 Impact factor: 2.367
Authors: L Smith-Thomas; J W Haycock; R Metcalfe; M Boulton; S Ellis; I G Rennie; P S Richardson; I Palmer; M A Parsons; S Mac Neil Journal: Curr Eye Res Date: 1998-08 Impact factor: 2.424
Authors: Sarah K Ohlemacher; Clara L Iglesias; Akshayalakshmi Sridhar; David M Gamm; Jason S Meyer Journal: Curr Protoc Stem Cell Biol Date: 2015-02-02
Authors: Roxanne H Croze; David E Buchholz; Monte J Radeke; William J Thi; Qirui Hu; Peter J Coffey; Dennis O Clegg Journal: Stem Cells Transl Med Date: 2014-07-28 Impact factor: 6.940
Authors: Elizabeth E Capowski; Kayvan Samimi; Steven J Mayerl; M Joseph Phillips; Isabel Pinilla; Sara E Howden; Jishnu Saha; Alex D Jansen; Kimberly L Edwards; Lindsey D Jager; Katherine Barlow; Rasa Valiauga; Zachary Erlichman; Anna Hagstrom; Divya Sinha; Valentin M Sluch; Xitiz Chamling; Donald J Zack; Melissa C Skala; David M Gamm Journal: Development Date: 2019-01-09 Impact factor: 6.868