Electrical stimulation combined with p27Kip1 inactivation drives proliferative neurogenic reprogramming of Mueller glia in the adult mouse retina

Mueller glial reprogramming studies demonstrate that mammalian Mueller glia can be induced to proliferate and/or engage in neural differentiation, as occurs naturally in teleost fish. A major objective is the identification of combined strategies that promote both robust proliferation and neurogenesis. These studies would benefit from a translatable screening platform that enables controlled perturbation, maintained tissue context and longitudinal analysis, such as 3D culture for first tier analysis of reprogramming strategies. Here, we validate a 3D retinal culture for Mueller glial reprogramming studies by recapitulating key signatures of an <i>in vivo</i> reprogramming paradigm. Next, we find that electrical stimulation (E-stim) as a tunable, extrinsic cue is sufficient to activate endogenous Ascl1 expression, indicating a state transition favorable for neurogenesis, while Mueller glia-specific <i>p27</i> ^<i>Kip1</i> inactivation promotes robust, prolonged proliferation. Utilization of the lineage-tracing proliferation-history reporter H3.1-iCOUNT enabled longitudinal proliferation analysis and assessment of reprogramming outcomes within the proliferative, Mueller-derived population. With this model, we find that E-stim and <i>p27</i> ^<i>Kip1</i> inactivation in combination (ESPI) increases proliferation, endogenous Ascl1 expression, and neurogenesis of Mueller-derived cells across modalities. Together, this work establishes a 3D culture framework for discovery of combinatorial reprogramming strategies within a proliferative context and identifies ESPI as an efficient approach to proliferative, neurogenic Mueller glial reprogramming.