USING NEXT GENERATION VIRUSES TO INTERROGATE IPSC-DERIVED NEURONS AND MICROGLIA

Traditional viral vectors adeno-associated virus (AAV) and lentivirus, have a minimal cargo capacity. Conversely BacMam, a double-stranded DNA insect virus, can carry up to 38 kilobases. This payload enables the use of large cell-type-specific promoters and large cargos. Our question was whether these promising vectors can be used to reliably express genes of interest in neurons and glial cells. We undertook this study using BrainXell’s iPSC-derived cortical glutamatergic neurons, astrocytes, and microglia. We transduced with next-generation baculovirus engineered for high expression. These viruses delivered genetically encoded biosensors as well as probes for mitochondria, lysosomes, omegasomes, cytoskeleton, nucleus, Golgi, and endosomes. We tested transduction on days 0, 1, 4, and 7 in the two-week maturation protocol from BrainXell. The viruses produced robust expression without obvious toxicity if used on day 4 after seeding or later. Indeed, in mature cultures, as little as 24 hours was needed for strong expression. Regardless of expression level, the probes correctly decorated the specific organelles. The introduction of cADDis, a cAMP sensor, made it possible to study endogenous receptor activation.

Ultimately, the neuroscientist wants to study co-cultures of many different cell types. Therefore, we wanted to determine if we could use cell-type-specific promoters in BacMam. We transduced immature iPSC-derived cortical glutamatergic neurons with a synapsin-driven mNeongreen. This produced no fluorescence until the cells matured ~14 days later, upon which they became bright green. Work is underway with other cell-type-specific promoters to be able to differentiate specific neuronal subpopulations such as astrocytes and microglia.