Scientists report on new studies demonstrating that human embryonic stem cell (hESC)-derived neurons implanted into experimental animals can make both excitatory and inhibitory synaptic connections with individual neurons, and also elicit post-synaptic currents. The work, by a team at University of Wisconsin–Madison provides further evidence that hESC-derived neurons can fully integrate into the brains of transplant recipients.
Jason P. Weicka, Ph.D., Yan Liua, Ph.D., and Su-Chun Zhang, Ph.D., used optogenetic technology to demonstrate that hESC-derived neurons are capable of complete synaptic integration with a pre-existing network both in vitro and in vivo, and can modulate the excitability of a network via synaptic output. They report their findings in PNAS, in a paper titled “Human embryonic stem cell-derived neurons adopt and regulate the activity of an established neural network.”
Considerable work has already shown that hESC- and iPSC-derived neuronal subtypes can correct behavior deficits when transplanted into animal models, and express basic functional properties such as action potential (AP) firing and synaptic currents. Studies on slices taken from the brains of transplant recipient animals have demonstrated that hESC- and iPSC-derived neurons demonstrate spontaneous postsynaptic currents, which are believed to be triggered by presynaptic transmitter release from host neurons.
“However, complete functional integration requires more complex physiological properties, including PSC-induced spiking, presynaptic outputs to surrounding neurons, and the ability to regulate the behavior of a pre-existing neural network,” the authors write. Generating this sort of data has been hampered by the technological difficulties associated with stimulating groups of neurons simultaneously.
To address this, the Wisconsin-Madison team used optogenetic targeting of hESC-derived neurons to test these capabilities in vitro and in vivo. They first plated hESC-derived neuroepithelial aggregates with cultures of embryonic mouse cortical neurons, which display a synchronized network activity known as bursting. While the hESC-derived neurons plated alone showed no bursting activity over weeks of culture, human neurons co-cultured with the mouse cortical neurons displayed prominent bursting activity. This feature was observed in some hESC-derived neurons after two weeks of co-culture, by the majority at four weeks, and by nearly all the neurons after 6–8 weeks.
Dual patch-clamp recordings in addition confirmed that the bursting activity in human neurons was a result of their functional integration with the existing mouse network, and not because they had generated a new human network. Interestingly, recordings indicated that although the mouse and human bursts occurred nearly simultaneously, the mouse bursts occurred about 50–70 ms before those of the human neurons.
In another series of tests the team delivered light pulses to demonstrate that activating the hESC-derived neurons in the co-cultures was sufficient to induce spiking in the human cells, and bursting behavior in the mouse neurons. The results were reproducible, with the light-induced APs in the hESC-derived neurons preceding bursting activity in the mouse cells by about 67–80 ms, and the bursting mimicking the spontaneous bursting activity. Moreover, light-induced bursting could be attributed to multiple hESC-derived neurons simultaneously triggering PSCs in mouse cells, the authors state, because in those mouse cells in which light stimulation did not induce bursting, multiple PSCs were triggered immediately after the light pulse.
The initial studies had in addition demonstrated that in co-cultures of hESC-derived neurons and mouse cortical neurons, spontaneous bursting could be eliminated by treatment with the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline- 2, 3-dione (CNQX). These findings, in addition to further studies evaluating electrical activity in neurons treated with CNQX, pixantrone, and the NMDA receptor antagonist D-2-amino-5-phosphonopentanoic acid (AP5), indicated that hESC-derived neurons regulated the mouse network excitability via excitatory (glutamatergic) and/or inhibitory (GABAergic) synaptic connections.
The team moved on to determine whether hESC-derived neurons could form functional presynaptic connections with mouse neurons in vivo, by implanting neuroepithelial aggregates infected with Syn-ChR2(H134R)-mCherry to the CA3 region of the hippocampus of two-month-old SCID mice. Histological examination of brain tissue confirmed that mCherry+ neurons had migrated through the CA3, CA1, and dentate gyrus regions. Evaluation of brain slices also showed that all the labeled cells displayed inward and outward voltage-gated currents as well as spiking in response to current injection, and most of the hESC-derived neurons demonstrated spontaneous PSCs. Importantly, mouse neurons recorded near mCherry+ processes demonstrated passive and active properties of pyramidal neurons, and generated trains of accommodating APs in response to current injection.
The authors claim that while further research is needed confirm whether a causal association between synaptic integration and behavioral changes observed after stem cell-derived neuronal transplantation, their data “definitively demonstrate that hESC-derived neurons are capable of complete synaptic integration with a pre-existing network both in vitro and in vivo, and can modulate the excitability of a network via synaptic output.” They also suggest that their optogenetics approach may have a broad utility investigating the physiological processes that occur following stem cell transplantation.