Restoration of Cortical Network after Ischemic Stroke

Abstract

Background: Transplanted neurons derived from stem cells have been proposed to improve function in animal models of human disease by various mechanisms such as neuronal replacement. Stem cell transplantation can improve behavioral recovery after stroke in animal models but whether stem cell-derived neurons become functionally integrated into stroke-injured brain circuitry by receiving functional synaptic inputs from the recipient’s brain and establishing efferent synaptic connections with host neural circuitry is unknown.

Aim: The main aim of the study was to investigate the synaptic inputs from the host brain to grafted cortical neurons derived from human induced pluripotent stem cells (iPSCs) after transplantation into stroke-injured rat cerebral cortex. Also, we explored whether grafted neurons also send widespread axonal projections to the host brain.

Materials and Methods: We used rat model of focal cortical stroke induced by occlusion of distal branch of middle cerebral artery. The rabies virus-based trans-synaptic tracing method and immunoelectron microscopy was used to identify afferent and efferent synaptic connections of grafted neurons. Electrophysiological in vivo recordings and patch-clamp recordings from acute brain slices were used to study electrical activity and network connections of grafted reprogrammed neurons. For the behavioral assessment the of a sensorimotor function we used cylinder test.

Results: We demonstrated that the grafted neurons receive direct synaptic inputs from neurons in different host brain areas located in a pattern similar to that of neurons projecting to the corresponding endogenous cortical neurons in the intact brain. The rabies virus–based transsynaptic tracing, showed that host neurons in the contralateral somatosensory cortex receive monosynaptic inputs from grafted neurons. Moreover, immunoelectron microscopy demonstrated myelination of the graft-derived axons in the corpus callosum and that their terminals form excitatory, glutamatergic synapses on host cortical neurons. Electrophysiological in vivo recordings from the cortical implants showed that physiological sensory stimuli can activate spontaneous activity in grafted neurons, indicating that at least some of the afferent inputs are functional. In agreement, we found that a portion of grafted neurons respond to photostimulation of virally transfected, channelrhodopsin-2-expressing thalamo-cortical axons in acute brain slices. We showed that the stroke-induced asymmetry in a sensorimotor function is reversed by transplantation. Light-induced inhibition of halorhodopsin-expressing, grafted neurons did not recreate the impairment, indicating that its reversal is not due to neuronal activity in the graft.

Conclusions: The present study demonstrates, for the first time, that the host brain regulates the activity of grafted neurons, providing strong evidence that transplanted human iPSC-derived cortical neurons can become incorporated into injured cortical circuitry. Our data indicate that activity in the grafted neurons, probably mediated through transcallosal connections to the contralateral hemisphere, is involved in maintaining normal motor function. This is an example of functional integration of efferent projections from grafted neurons into the stroke-affected brain’s neural circuitry, which raises the possibility that such repair might be achievable also in humans affected by stroke.