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Optogenetically engineered human neural progenitors (hNPs) are viewed as promising tools in regenerative neuroscience because they allow the testing of the ability of hNPs to integrate within nervous system of an appropriate host not only structurally, but also functionally based on the responses of their differentiated progenies to light. Here, we transduced H9 embryonic stem cell-derived hNPs with a lentivirus harboring human channelrhodopsin (hChR2) and differentiated them into a forebrain lineage. We extensively characterized the fate and optogenetic functionality of hChR2-hNPs  in vitro  with electrophysiology and immunocytochemistry. We also explored whether the  in vivo  phenotype of ChR2-hNPs conforms to  in vitro  observations by grafting them into the frontal neocortex of rodents and analyzing their survival and neuronal differentiation. Human ChR2-hNPs acquired neuronal phenotypes (TUJ1, MAP2, SMI-312, and synapsin 1 immunoreactivity)  in vitro  after an average of 70 days of coculturing with CD1 astrocytes and progressively displayed both inhibitory and excitatory neurotransmitter signatures by immunocytochemistry and whole-cell patch clamp recording. Three months after transplantation into motor cortex of naïve or injured mice, 60–70% of hChR2-hNPs at the transplantation site expressed TUJ1 and had neuronal cytologies, whereas 60% of cells also expressed ChR2. Transplant-derived neurons extended axons through major commissural and descending tracts and issued synaptophysin +  terminals in the claustrum, endopiriform area, and corresponding insular and piriform cortices. There was no apparent difference in engraftment, differentiation, or connectivity patterns between injured and sham subjects. Same trends were observed in a second rodent host, i.e. rat, where we employed longer survival times and found that the majority of grafted hChR2-hNPs differentiated into GABAergic neurons that established dense terminal fields and innervated mostly dendritic profiles in host cortical neurons. In physiological experiments, human ChR2 +  neurons in culture generated spontaneous action potentials (APs) 100–170 days into differentiation and their firing activity was consistently driven by optical stimulation. Stimulation generated glutamatergic and GABAergic postsynaptic activity in neighboring ChR2 -  cells, evidence that hChR2-hNP-derived neurons had established functional synaptic connections with other neurons in culture. Light stimulation of hChR2-hNP transplants  in vivo  generated complicated results, in part because of the variable response of the transplants themselves. Our findings show that we can successfully derive hNPs with optogenetic properties that are fully transferrable to their differentiated neuronal progenies. We also show that these progenies have substantial neurotransmitter plasticity  in vitro , whereas  in vivo  they mostly differentiate into inhibitory GABAergic neurons. Furthermore, neurons derived from hNPs have the capacity of establishing functional synapses with postsynaptic neurons  in vitro , but this outcome is technically challenging to explore  in vivo . We propose that optogenetically endowed hNPs hold great promise as tools to explore  de novo  circuit formation in the brain and, in the future, perhaps launch a new generation of neuromodulatory therapies.

plos one

Optogenetically transduced human ES cell-derived neural progenitors and their neuronal progenies: Phenotypic characterization and responses to optical stimulation