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

Abstract

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 ...

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