title: Use of a synthetic xeno-free culture substrate for induced pluripotent stem cell induction and retinal differentiation.
abstract: The purpose of this study was to determine whether a proprietary xeno-free synthetic culture surface could be used to aid in the production and subsequent retinal-specific differentiation of clinical-grade induced pluripotent stem cells (iPSCs). iPSCs were generated using adult somatic cells via infection with either a single cre-excisable lentiviral vector or four separate nonintegrating Sendai viruses driving expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC. Retinal precursor cells were derived via targeted differentiation of iPSCs with exogenous delivery of dkk-1, noggin, insulin-like growth factor-1, basic fibroblast growth factor, acidic fibroblast growth factor, and DAPT. Phase contrast microscopy, immunocytochemistry, hematoxylin and eosin staining, and reverse transcription-polymerase chain reaction were used to determine reprogramming efficiency, pluripotency, and fate of undifferentiated and differentiated iPSCs. Following viral transduction, cells underwent prototypical morphological changes resulting in the formation of iPSC colonies large enough for manual isolation/passage at 3-4 weeks postinfection. Both normal and disease-specific iPSCs expressed markers of pluripotency and, following transplantation into immune-compromised mice, formed teratomas containing tissue comprising all three germ layers. When subjected to our established retinal differentiation protocol, a significant proportion of the xeno-free substrate-derived cells expressed retinal cell markers, the number of which did not significantly differ from that derived on traditional extracellular matrix-coated dishes. Synthetic cell culture substrates provide a useful surface for the xeno-free production, culture, and differentiation of adult somatic cell-derived iPSCs. These findings demonstrate the potential utility of these surfaces for the production of clinical-grade retinal neurons for transplantation and induction of retinal regeneration.
authors.affiliation: Department of Ophthalmology, University of Iowa, Iowa City, IA, USA. budd-tucker@uiowa.edu
authors.forename: Budd A
authors.initials: BA
authors.lastname: Tucker
authors.forename: Kristin R
authors.initials: KR
authors.lastname: Anfinson
authors.forename: Robert F
authors.initials: RF
authors.lastname: Mullins
authors.forename: Edwin M
authors.initials: EM
authors.lastname: Stone
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Young
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D003470:Culture Media; D005347:Fibroblasts / Q000502:physiology; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism / Q000502:physiology*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D057507:Mice, 129 Strain; D016513:Mice, SCID; D000071317:Nanog Homeobox Protein; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012162:Retinal Degeneration / Q000473:pathology; D055351:Retinal Neurons; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D029082:Sendai virus / Q000235:genetics; D012867:Skin / Q000473:pathology; D013499:Surface Properties
chemical_list: D000943:Antigens, Differentiation; D003470:Culture Media; D018398:Homeodomain Proteins; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C489427:MYC protein, human; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D016271:Proto-Oncogene Proteins c-myc; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors