Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Nrl-/- Retinal Transcriptomes

Neural precursor cells (NPCs) tend to aggregate and develop into three-dimensional (3D) spheres, which in return help maintain the stemness of the cells. This close relationship between spherical environment and cell stemness direct us to assume that 3D spheres of astrocytes may facilitate the acquiring of stem cell-like features and generate sufficient seed cells for regeneration of neurons. Primary astrocytes (ASTs) were isolated from the cerebra of postnatal day 1 to day 2 C57BL/6 mouse. ASTs were loaded onto the agarose-coated dishes and cultured to induce the formation of AST spheres. The status of the ASTs over time in cell spheres was characterized by immunochemistry, flow-cytometry, western blotting, and RNA-sequencing. The potency of AST-derived stem cells (A-iSCs) in repairing neural injuries was evaluated in mouse models of middle cerebral artery occlusion (MCAO), spinal cord injury (SCI), and peripheral nerve injury (PNI). In vitro results confirmed that mouse astrocytes cultured on agarose surfaces spontaneously formed cell spheres and exhibited molecular features similar to stem cells, capable of further differentiating into neurons particularly and forming functional synaptic networks with synchronous burst activities. RNA-sequencing results revealed the similarity of A-iSCs to NPCs in global gene expression profiles. It was observed that the transplanted A-iSCs expressed a series of markers for neural differentiation, such as NeuN, Tuj1, and Map2, indicating the conversion of the transplanted A-iSCs into neurons in these scenarios. We also found that the injured mice injected with A-iSCs exhibited significant improvements in sensorimotor functions over 8 weeks compared with the sham and control mice. Taken together, mouse astrocytes form cell spheres on agarose surfaces and acquire stem cell-associated features, and the derived A-iSCs possess the capacity to differentiate into neurons and facilitate the regeneration of damaged nerves across the nervous system. To elucidate the mechanism involved in the induction of ASTs in 3D cell spheres, global gene expression profile of ASTs (0 day incubation, negative control) and A-iSCs (5 days incubation) were characterized by RNA-sequencing, with the profiles of brain-derived P56-NPCs and P0-NPCs as positive controls. Three replicates per sample. ***Submitters declare that the raw data has been deleted and can not be recovered***