In Vitro Assay for the Detection of Network Connectivity in Embryonic Stem Cell-Derived Cultures

Stem cell transplantation holds great promise as a repair strategy following spinal cord injury. Embryonic stem cell (ESC) transplantation therapies have elicited encouraging though limited improvement in motor and sensory function with the use of heterogeneous mixtures of spinal cord neural progenitors and ESCs. Recently, transgenic lines of ESCs have been developed to allow for purification of specific candidate populations prior to transplantation, but the functional network connectivity of these populations and its relationship to recovery is difficult to examine with current technological limitations. In this study, we combine an ESC differentiation protocol, multi-electrode arrays (MEAs), and previously developed neuronal connectivity detection algorithms to develop an in vitro high-throughput assay of network connectivity in ESC-derived populations of neurons. Neuronal aggregation results in more consistent detection of individual neuronal activity than dissociated cultures. Both aggregated and dissociated culture types exhibited synchronized bursting behaviors at days 17 and 18 on MEAs, and thousands of statistically significance functional connections were detected in both culture types. Aggregate cultures, however, demonstrate a tight linear relationship between the inter-neuron distance of neuronal pairs and the time delay of the neuronal pair functional connections, whereas dissociated cultures do not. These results suggest that ESC-derived aggregated cultures may reflect some of the spatiotemporal connectivity characteristics of in vivo tissue and prove to be useful models of investigating potentially therapeutic populations of ESC-derived neurons in vitro.