Pro-inflammatory cytokines during human RGC differentiation facilitate neuritogenesis and regeneration

Abstract

Purpose : Microglia provide homeostatic support during development and function of the retina. The homeostatic regulation involves glia-neuron interactions through microglia-elaborated cytokines and growth factors. Here, we have examined whether IL1β and TNFα influence human retinal ganglion cells (hRGCs) differentiation and axon growth and guidance.

Methods : RGCs were differentiation from human ES ^Brn3b-tdT cells (Sluch et al., 2017) using our stage-specific chemically defined protocol that directly differentiate pluripotent cells into RGCs by recapitulate developmental mechanism (Teotia et al., 2017). The differentiation was initially carried out in the presence and absence of IL1β and TNFα cytokines. Co-culture with BV2 cells were carried out to corroborate results obtained in response to cytokines. The effects of RGC differentiation, neurite morphology, axon length and expression of growth and guidance molecules were examined. Regeneration was examined using our microfluidic model of axon regeneration (Teotia et al., 2019).

Results : We observed that the differentiation of hRGC ^Brn3b-tdT was similar in terms of the number of nascent (tdT⁺β-tubulin⁺cells) and differentiated (tdT⁺ and PAX6⁺ cells) RGCs between cytokine and control groups except in the former the number of matured hRGCs (tdT⁺THY1⁺ cells; p>0.05) was comparatively higher. However, the morphology of hRGC^Brn3b-tdT was significantly impacted by cytokines, as the length of axons (SMI32⁺TAU1⁺ neurites; p>0.001) were significantly longer in the cytokine groups versus controls. Correspondingly, the expression of axon growth genes such as GAP43 (p> 0.03), KLF6 (p> 0.01), and SOX11 (p> 0.01) and genes encoding guidance receptors such as DCC (p> 0.004), and EPHB2 (p> 0.02) was significantly higher in cytokine groups, compared to controls. The cytokines influenced RGC^Brn3b-tdT function as well, facilitating a significantly high spontaneous action potential (P>0.004), compared to control, in multielectrode array (MEA) analyses. The positive effect of cytokines on axon growth was also reflected in improved RGC^Brn3b-tdT axon regeneration following axotomy, compared to controls.

Conclusions : Our results posit a positive role for proinflammatory cytokines during early stages of human RGC development. They may facilitate axon growth and guidance during differentiation and their cellular action can be recapitulated to sustain optic nerve regeneration.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.