Controlling donor and newborn neuron migration and maturation in the eye through microenvironment engineering.

Ongoing cell therapy trials have demonstrated the need for precision control of donor cell behavior within the recipient tissue. We present a methodology to guide stem cell-derived and endogenously regenerated neurons by engineering the microenvironment. Being an "approachable part of the brain," the eye provides a unique opportunity to study neuron fate and function within the central nervous system. Here, we focused on retinal ganglion cells (RGCs)-the neurons in the retina are irreversibly lost in glaucoma and other optic neuropathies but can potentially be replaced through transplantation or reprogramming. One of the significant barriers to successful RGC integration into the existing mature retinal circuitry is cell migration toward their natural position in the retina. Our in silico analysis of the single-cell transcriptome of the developing human retina identified six receptor-ligand candidates, which were tested in functional in vitro assays for their ability to guide human stem cell-derived RGCs. We used our lead molecule, SDF1, to engineer an artificial gradient in the retina, which led to a 2.7-fold increase in donor RGC migration into the ganglion cell layer (GCL) and a 3.3-fold increase in the displacement of newborn RGCs out of the inner nuclear layer. Only donor RGCs that migrated into the GCL were found to express mature RGC markers, indicating the importance of proper structure integration. Together, these results describe an "in silico-in vitro-in vivo" framework for identifying, selecting, and applying soluble ligands to control donor cell function after transplantation.

mTor signaling is required for the formation of proliferating Müller glia-derived progenitor cells in the chick retina.

We investigate the roles of mTor signaling in the formation of Müller glia-derived progenitor cells (MGPCs) in the chick retina. During embryonic development, pS6 (a readout of active mTor signaling) is present in early-stage retinal progenitors, differentiating amacrine and ganglion cells, and late-stage progenitors or maturing Müller glia. By contrast, pS6 is present at low levels in a few scattered cell types in mature, healthy retina. Following retinal damage, in which MGPCs are known to form, mTor signaling is rapidly activated in Müller glia. Inhibition of mTor in damaged retinas prevented the accumulation of pS6 in Müller glia and reduced numbers of proliferating MGPCs. Inhibition of mTor had no effect on MAPK signaling or on upregulation of the stem cell factor Klf4, whereas Pax6 upregulation was significantly reduced. Inhibition of mTor potently blocked the MGPC-promoting effects of Hedgehog, Wnt and glucocorticoid signaling in damaged retinas. In the absence of retinal damage, insulin, IGF1 and FGF2 induced pS6 in Müller glia, and this was blocked by mTor inhibitor. In FGF2-treated retinas, in which MGPCs are known to form, inhibition of mTor blocked the accumulation of pS6, the upregulation of Pax6 and the formation of proliferating MGPCs. We conclude that mTor signaling is required, but not sufficient, to stimulate Müller glia to give rise to proliferating progenitors, and the network of signaling pathways that drive the formation of MGPCs requires activation of mTor.

Reactive microglia and macrophage facilitate the formation of Müller glia-derived retinal progenitors.

In retinas where Müller glia have been stimulated to become progenitor cells, reactive microglia are always present. Thus, we investigated how the activation or ablation of microglia/macrophage influences the formation of Müller glia-derived progenitor cells (MGPCs) in the retina in vivo. Intraocular injections of the Interleukin-6 (IL6) stimulated the reactivity of microglia/macrophage, whereas other types of retinal glia appear largely unaffected. In acutely damaged retinas where all of the retinal microglia/macrophage were ablated, the formation of proliferating MGPCs was greatly diminished. With the microglia ablated in damaged retinas, levels of Notch and related genes were unchanged or increased, whereas levels of ascl1a, TNFα, IL1ß, complement component 3 (C3) and C3a receptor were significantly reduced. In the absence of retinal damage, the combination of insulin and Fibroblast growth factor 2 (FGF2) failed to stimulate the formation of MGPCs when the microglia/macrophage were ablated. In addition, intraocular injections of IL6 and FGF2 stimulated the formation of MGPCs in the absence of retinal damage, and this generation of MGPCs was blocked when the microglia/macrophage were absent. We conclude that the activation of microglia and/or infiltrating macrophage contributes to the formation of proliferating MGPCs, and these effects may be mediated by components of the complement system and inflammatory cytokines.