Activity in grafted human iPS cell-derived cortical neurons integrated in stroke-injured rat brain regulates motor behavior

Abstract

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 is poorly understood. Here we show that intracortically grafted human induced pluripotent stem (iPS) cell-derived cortical neurons send widespread axonal projections to both hemispheres of rats with ischemic lesions in the cerebral cortex. Using rabies virus-based transsynaptic tracing, we find that at 6 mo after transplantation, host neurons in the contralateral somatosensory cortex receive monosynaptic inputs from grafted neurons. Immunoelectron microscopy demonstrates myelination of the graft-derived axons in the corpus callosum and that their terminals form excitatory, glutamatergic synapses on host cortical neurons. We show that the stroke-induced asymmetry in a sensorimotor (cylinder) test is reversed by transplantation. Light-induced inhibition of halorhodopsin-expressing, grafted neurons does not recreate the impairment, indicating that its reversal is not due to neuronal activity in the graft. However, we find bilateral decrease of motor performance in the cylinder test after light-induced inhibition of either grafted or endogenous halorhodopsin-expressing cortical neurons, located in the same area, and after inhibition of endogenous halorhodopsin-expressing cortical neurons by exposure of their axons to light on the contralateral side. 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.

Keywords: cerebral cortex; iPS cells; optogenetics; stroke; transplantation.

Fig. 1.

Cellular composition of cortically fated human lt-NES cell–derived grafts in stroke-injured rat somatosensory cortex. (A–C) Confocal immunohistochemical images showing mature neurons (A, NeuN), glutamatergic neurons (B, KGA), and neuronal progenitors (C, DCX) in the transplant core (human cells are STEM101+). (D–G) Expression of markers characteristic of neurons in upper (D, Brn2 and E, Satb2) and deep (F, Tbr1 and G, Ctip2) cortical layers in the human cells. Arrows indicate colocalization. (Scale bar, 20 μm.)

Fig. 2.

Intracortical grafts of human lt-NES cell–derived cortical neurons project extensively in stroke-injured rat brain. (A) Location of illustrated areas. (B–D) Fibers immunoreactive for human cytoplasmic marker STEM121 in the transplant core (B), corpus callosum (C), and peri-infarct area (D). (Scale bar, 200 μm.)

Fig. 3.

Grafted human lt-NES cell–derived cortical neurons establish monosynaptic connections with host neurons in the contralateral cortex of stroke-injured rats. (A) Brain slice showing GFP+ grafted neurons in ipsilateral and mCherry+ host neurons transduced by rabies virus in the contralateral somatosensory cortex. (B) Confocal images of mCherry+, GFP+ grafted cell expressing the corticocallosal projection neuron marker Satb2 (gray) in the transplant core. Nuclear staining (Hoechst, blue) is included in the merged panel. (Scale bar in B, 20 μm.)

Fig. 4.

Axons originating in grafted human lt-NES cell–derived cortical neurons become myelinated in stroke-injured rat brain. Graft-derived axons (marked in green) ensheathed by myelin in the (A) ipsilateral somatosensory cortex, (B) corpus callosum (CC), and (C) contralateral somatosensory cortex. (Scale bar, 0.2 μm.)

Fig. 5.

Axon myelination occurs at different stages at 6 mo posttransplantation, and myelin partly originates from human-derived oligodendrocytes. (A–C) Graft-derived axons in the contralateral somatosensory cortex. (A) Initial and (B) intermediate stages of myelination. (C) Axons with compact myelin sheaths. m: mitochondria. (Scale bar, 0.2 μm.) (D) Confocal image of an axon ensheathed by human myelin close to the transplant, as evidenced by colocalization of MBP (red) and human mitochondrial marker (green). (Scale bar, 5 μm.) (E) Confocal image of graft-derived fibers expressing the human cytoplasmic marker STEM121 (green) colocalizing with MBP (red). (Scale bar, 10 μm.)

Fig. 6.

Grafted human lt-NES cell–derived cortical neurons establish asymmetric synapses with host neurons in the contralateral somatosensory cortex of stroke-injured rats. Synapses with continuous (A) and perforated (B) postsynaptic densities (red lines) between host dendritic spines (ds, brown) and grafted GFP+/DAB+ presynaptic axon terminals (at, green). (Scale bar, 0.2 μm.)

Fig. 7.

Activity in grafts of human lt-NES cell–derived cortical neurons is not responsible for reversal of stroke-induced sensorimotor deficit in the cylinder test but regulates motor function bilaterally similar to endogenous cortical neurons. (A′) Percentage the left limb touches in sham surgery + vehicle injection (sham+Veh; n = 5), dMCAO+Veh (n = 6), and dMCAO + transplantation of cortically fated lt-NES cells (dMCAO+Cells) (n = 11) groups in the cylinder test (*P < 0.05 vs. dMCAO+Veh). (A´´) Percentage the left limb touches in rats transplanted with GFP (n = 5) or eNpHR3.0-YFP lt-NES (n = 6) cells with and without light stimulation (P > 0.05). (A´´´) Total number of touches and (A´´´´) number of touches of individual limbs in animals with eNpHR3.0 lt-NES cell transplants with and without light stimulation (*P < 0.05 light on vs. light off). (B′) Percentage the left limb touches (P > 0.05 vs. light off) and (B´´) total number of touches of individual limbs with and without light stimulation in the ipsilateral or contralateral hemisphere (*P < 0.05 vs. light off; n = 6) in intact animals injected with eNpHR3.0 virus in the somatosensory cortex. Means ± SEM.