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Nat Protoc. 2016 Oct;11(10):1955-1976. doi: 10.1038/nprot.2016.109. Epub 2016 Sep 15.

Reprogramming of mouse retinal neurons and standardized quantification of their differentiation in 3D retinal cultures.

Author information

1
Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
2
Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, USA.
3
Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.

Abstract

Postmitotic differentiated neurons are among the most difficult cells to reprogram into induced pluripotent stem cells (iPSCs) because they have poor viability when cultured as dissociated cells. To overcome this, other protocols have required the inactivation of the p53 tumor suppressor to reprogram postmitotic neurons, which can result in tumorigenesis of the cells. We describe a method that does not require p53 inactivation but induces reprogramming in retinal cells from reprogrammable mice grown in aggregates with wild-type mouse retinal cells. After the first 10 d of reprogramming, the aggregates are then dispersed and plated on irradiated feeder cells to propagate and isolate individual iPSC clones. The reprogramming efficiency of different neuronal populations at any stage of development can be quantified using this protocol. Reprogramming retinal neurons using this protocol will take 56 d, and these retina-derived iPSCs can undergo retinal differentiation to produce retinae in 34 d. In addition, we describe a quantitative assessment of retinal differentiation from these neuron-derived iPSCs called STEM-RET. The procedure quantifies eye field specification, optic cup formation and retinal differentiation in 3D cultures using molecular, cellular and morphological criteria. An advanced level of cell culture experience is required to carry out this protocol.

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