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

Developers

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

Description of the technology

This is a technology for reprogramming of mouse retinal neurons to generate pluripotent stem cells. 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 technology use the inactivation of the p53 tumor suppressor to reprogram postmitotic neurons, which can result in tumorigenesis of the cells.

However, this technology 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 the protocol according to this technology. 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, the protocol describes 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.

Practical application

The technology for reprogramming retinal neurons and quantifying their retinal differentiation allows investigators to produce stem cells of diverse lineages from the reprogrammable mouse and to identify the best source of stem cells for studying retinal development and disease, and potentially for cell-based therapies for human retinopathies.

Although STEM-RET was developed using murine stem cells, it could be readily adapted for human stem cells using a longer culture period. The cell surface markers would have to be used to perform similar studies with human retina. STEM-RET protocol could be used for quantitative comparisons of different stem cell lines to identify those that are the most efficient at making photoreceptors in culture for subsequent transplantation or disease modeling.

STEM-RET protocol provides a clear, standardized method for quantifying retinogenesis in vitro from different stem cell lineages regardless of origin.

Laboratories

• Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis (USA)
• Department of Ophthalmology, University of Tennessee Health Science Center, Memphis (USA)
• Howard Hughes Medical Institute, Chevy Chase (USA)

Links

Publications

• Hiler, D.J. et al. «Reprogramming of mouse retinal neurons and standardized quantification of their differentiation in 3D retinal cultures." 11 Nature Protocols (2016): 1955–1976.
• Hiler, D. et al. «Quantification of retinogenesis in 3D cultures reveals epigenetic memory and higher efficiency in iPSCs derived from rod photoreceptors." 17 Cell Stem Cell (2015): 101–115.