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

Nature Protocols volume 11, pages 1955–1976 (2016)Cite this article

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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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Figure 1: Diagram of reprogramming protocol using retinal pellet explants.
Figure 2: STEM-RET differentiation protocol with representative retinal spheres.
Figure 3: Preparation of a retinal pellet explant.
Figure 4: Representative micrographs of retinal features used in RDEM scoring.
Figure 5: Representative micrographs of immunofluorescence used in RDIF scoring.

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Acknowledgements

We thank St. Jude Shared Resource Facilities: Flow Cytometry, Light Microscopy, Electon Microscopy, Veterinary Pathology, Cytogenetics and Biomedical Communications for their contributions to data collection and analysis. This work was supported, in part, by a Cancer Center Support grant (CA21765 to M.A.D.) from the National Cancer Institute (NCI), grants to M.A.D. from the NIH (EY014867, EY018599 and CA168875) and the American Lebanese Syrian Associated Charities (ALSAC). M.A.D. was also supported by a grant from Alex's Lemonade Stand Foundation for Childhood Cancer. R.N.E. was supported by grant RO1CA20525 from the NCI. P.A.C. was supported by an NCI training grant (T32CA009657).

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  1. Daniel J Hiler and Marie E Barabas: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Daniel J Hiler, Marie E Barabas, Lyra M Griffiths & Michael A Dyer

  2. Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, USA

    Michael A Dyer

  3. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    Michael A Dyer

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  1. Daniel J Hiler
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Contributions

D.J.H. and M.A.D. developed the concepts, designed and performed experiments, analyzed data and developed the STEM-RET analysis. D.J.H. and M.E.B. wrote the paper, designed figures and performed data analysis. M.A.D. and L.M.G. supervised the project.

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Correspondence to Michael A Dyer.

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The authors declare no competing financial interests.

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Hiler, D., Barabas, M., Griffiths, L. et al. Reprogramming of mouse retinal neurons and standardized quantification of their differentiation in 3D retinal cultures. Nat Protoc 11, 1955–1976 (2016). https://doi.org/10.1038/nprot.2016.109

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