(171h) Kinetic Characterization of Proliferation and Dedifferentiation of Müller Cells

Kinetic Characterization of Proliferation and Dedifferentiation of Müller Cells

Alonzo D. Cook, Chris Burns, Ryan Gillis, Steven Christiansen

               Approximately 11 million Americans suffer from dry age-related macular degeneration (dry AMD), an eye disease that occurs in the macula of the eye when retinal pigment epithelial (RPE) cells experience oxidative stress and die. In several animals (including rats, zebra fish, and chicks), photoreceptor regeneration is possible with the appropriate stimulus. Müller cells have been shown to be the critical cell in photoreceptor regeneration.  In zebra fish and chicks, Müller cells dedifferentiate to a proliferating progenitor cell when photoreceptors are damaged.   These progenitor cells are then transported to their final area in retinal tissue where they differentiate to photoreceptors and integrate into the retinal tissue. Activation of cellular signaling pathways (Notch, MAPK, and others) is integral to this process. On each level of regeneration (dedifferentiation, proliferation, differentiation, migration and integration), these signaling pathways guide cell behavior, producing new photoreceptors that replace dead or damaged photoreceptors.  A therapy guided by this knowledge could replace more intrusive methods to potentially cure dry AMD.

               Dedifferentiation and differentiation of Müller cells is a potential option for curing dry AMD. The therapy would involve inducing Müller cells to transform into the needed photoreceptors. This would likely involve initially dedifferentiating the Müller cell into a progenitor state and then inducing the progenitor cells to become photoreceptors.  Dedifferentiation is the process by which a cell that has already developed into a fully functioning adult cell returns back to a previous state. The dedifferentiation step has been qualitatively observed in vitro.  Determination of the proliferation kinetics was performed in-house by isolating Müller cells and obtaining time-resolved cell counts. A Wistar albino rat eye was dissected and the retina was removed, dissociated and cultured. The Müller cells were isolated and divided into several different 25 mL flasks. An initial cell concentration was measured with a microfluidic cell counter. At 12 hour time intervals, a flask was removed and cell counts were taken.

               Assuming a 1^st order model of proliferation dependent on cell concentration, the rate constant was found to be 0.09/hr for cells cultured with fetal bovine serum. This rate constant dropped to 0.03/hr when fetal bovine serum was removed from the growth media. This value matches data reported by Limb¹ and is similar to the value derived from data reported by Zhao².  The shape of the cells was observed to shift to a dendritic neuronal shape. The dedifferentiated nature of the cells was confirmed by immunocytochemistry, observed qualitatively in the cells used in the proliferation experiments, and quantitatively described by protein ratios according to the method of Goldman, et al³.  The concentration of proteins typical of photoreceptor progenitor cells was assumed to be proportional to the number of cells, and the rate constant for dedifferentiation was determined to be 0.35/hr. This is significant because when combined with proliferation kinetic data, it is clear that dedifferentiation occurs much faster than proliferation.

               This project will continue by repeating the proliferation kinetic experiments and attempting to determine the rate constants for differentiation into photoreceptors. Also the model will be modified to handle non-first order rates. Eventually, once the system is defined, the model will allow the optimal design of therapies for retinal degeneration.

References

1. Limb, Astrid; et al. Differences between the neurogenic and proliferative abilities of Müller Glia with stem cell characteristics and the ciliary epithelium from the adult human eye. Exp Eye Res. 93(6): 852-61. Dec 2011.

2. Zhao. Induction of Retinal Progenitors and Neurons from Mammalian Müller Glia under Defined Conditions. J. Bio. Chem. Feb 2014.

3. Goldman, Daniel; et. al. Ascl1a regulates Müller Glia dedifferentiation and retina regeneration via a Lin-28-dependent, let-7 miRNA signaling pathway. Nat Cell Bio. 12(11): 1101-7. Nov 2010.