Abstract
Neuronal degeneration and the deterioration of neuronal communication lie at the origin of many neuronal disorders, and there have been major efforts to develop cell replacement therapies for treating such diseases. One challenge, however, is that differentiated cells are challenging to transplant due to their sensitivity both to being uprooted from their cell culture growth support and to shear forces inherent in the implantation process. Here, we describe an approach to address these problems. We demonstrate that rat hippocampal neurons can be grown on colloidal particles or beads, matured and even transfected in vitro, and subsequently transplanted while adhered to the beads into the young adult rat hippocampus. The transplanted cells have a 76% cell survival rate one week post-surgery. At this time, most transplanted neurons have left their beads and elaborated long processes, similar to the host neurons. Additionally, the transplanted cells distribute uniformly across the host hippocampus. Expression of a fluorescent protein and the light-gated glutamate receptor in the transplanted neurons enabled them to be driven to fire by remote optical control. At 1-2 weeks after transplantation, calcium imaging of host brain slice shows that optical excitation of the transplanted neurons elicits activity in nearby host neurons, indicating the formation of functional transplant-host synaptic connections. After 6 months, the transplanted cell survival and overall cell distribution remained unchanged, suggesting that cells are functionally integrated. This approach, which could be extended to other cell classes such as neural stem cells and other regions of the brain, offers promising prospects for neuronal circuit repair via transplantation of in vitro differentiated, genetically engineered neurons.
Highlights
Dysfunctions in synaptic transmission and degeneration of specific classes of neurons are at the origin of many neurological disorders [1,2,3,4,5,6,7,8]
Late embryonic stage (E18) hippocampal neurons were seeded on poly-L-lysine (PLL) coated beads using standard techniques developed for conventional 2D cultures [38,39] and adapted for 3D supports [36]
Young hippocampal cultures are poor in glia cells, we restored the glial growth factors known to contribute to neuronal development [40] with conditioned media from glial feeder cell cultures
Summary
Dysfunctions in synaptic transmission and degeneration of specific classes of neurons are at the origin of many neurological disorders [1,2,3,4,5,6,7,8]. The limited capacity of the mammalian central nervous system for self-repair makes cell transplantation an attractive approach to replace cells in damaged areas of the brain. The early signs of success of neural tissue grafts in animal models for disorders such as stroke [9,10], Huntington’s disease [11], brain lesion [12], and Parkinson disease [13,14] have made cell replacement therapy a highly promising clinical approach. Homotopic transplantation of normal embryonic neurons into the striatum of Huntington’s disease and Parkinson disease animal models [16,17,18], and into the hippocampus in models of temporal lobe epilepsy [19], appear to lead to cell survival and functional integration. The transplanted neurons remain within the injection area, limiting the reach of the functional repair
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