No abstract

We describe a technique for rapid labeling of a large number of cells in the nervous system with many different colors. By delivering lipophilic dye-coated particles to neuronal preparations with a "gene gun," individual neurons and glia whose membranes are contacted by the particles are quickly labeled. Using particles that are each coated with different combinations of various lipophilic dyes, many cells within a complex neuronal network can be simultaneously labeled with a wide variety of colors. This approach is most effective in living material but also labels previously fixed material. In living material, labeled neurons continue to show normal synaptic responses and undergo dendritic remodeling. This technique is thus useful for studying structural plasticity of neuronal circuits in living preparations. In addition, the Golgi-like labeling of neurons with many different colors provides a novel way to study neuronal connectivity.

Considerable progress has been made in testing stem cell–derived retinal pigment epithelium (RPE) as a potential therapy for age-related macular degeneration (AMD). However, the recent reports of oncogenic mutations in induced pluripotent stem cells (iPSCs) underlie the need for robust manufacturing and functional validation of clinical-grade iPSC-derived RPE before transplantation. Here, we developed oncogenic mutation-free clinical-grade iPSCs from three AMD patients and differentiated them into clinical-grade iPSC-RPE patches on biodegradable scaffolds. Functional validation of clinical-grade iPSC-RPE patches revealed specific features that distinguished transplantable from nontransplantable patches. Compared to RPE cells in suspension, our biodegradable scaffold approach improved integration and functionality of RPE patches in rats and in a porcine laser-induced RPE injury model that mimics AMD-like eye conditions. Our results suggest that the in vitro and in vivo preclinical functional validation of iPSC-RPE patches developed here might ultimately be useful for evaluation and optimization of autologous iPSC-based therapies.

We demonstrate that within the intact and spontaneously active retina, dendritic processes of ganglion cells exhibit rapid and extensive movements during the period of synaptogenesis. Marked restructuring occurs in seconds, but structural changes are relatively balanced across the dendritic arbor, maintaining overall arbor size and complexity over hours. Dendritic motility is regulated by spontaneous glutamatergic transmission. Both the rate and extent of the movements are decreased by antagonists to NMDA and non-NMDA glutamate receptors but are unaffected by tetrodotoxin, a sodium channel blocker. The dendritic movements are actin dependent and are controlled by the Rho family of small GTPases. Transfection of dominant-negative and constitutively active mutants into ganglion cells showed that Rac and Rho exert reciprocal effects on motility. We suggest that the Rho family of small GTPases could integrate activity-dependent and -independent signals from afferents, thereby adjusting target motility and maximizing the chance for initial contact and subsequent synaptogenesis.