To trace host to graft connectivity of transplanted hESC der

To trace host-to-graft connectivity of transplanted hESC-derived neurons, we next generated hESC lines that stably expressed the tracing and control constructs (Figures 2A and S2A). The engineered hESCs were differentiated using a protocol that results in a high yield of authentic and functional ventral midbrain patterned DA neurons (Kirkeby et al., 2012). The midbrain-patterned salvinorin a cost (Figures S2C–S2F) were transplanted into a 6-hydroxydopamine (6-OHDA)-lesion model of PD, with athymic, nude rats as hosts (n = 8 per group). At 6 weeks post-transplantation, two rats per group were injected with ΔG-rabies within and adjacent to the graft core and perfused 7 days later. Remaining animals were left for their grafts to mature for 6 months, as detailed in the experimental timeline (Figure S2B). At this time point, the grafts were rich in DA neurons (Figures S2G and S2H). Based on quantifications of mCherry, GFP and tyrosine hydroxylase (TH) expression (Figure S2I), we could determine that 44.7% ± 15.7% of the starter neurons expressed TH. We also stained for serotonergic neurons and found only a small number (2.1% ± 0.4%) of the starter neurons expressed 5-HT (Figure S2J). Already 6 weeks after transplantation, grafts were readily detectable using GFP immunostaining (Figure 2B), and we observed that the majority of GFP+ grafted neurons were infected with ΔG-rabies (Figures 2C–2D′). Interestingly, mCherry+ neurons that did not co-express GFP (thus representing host cells that had made synaptic contacts with the transplanted neurons) could be observed adjacent to the graft core (Figure 2D″). In areas distal to the transplant, we observed mCherry+/GFP− host neurons within the prefrontal and sensorimotor cortices (Figures 2E and 2I), the thalamus (Figures 2F and 2I), and a few as far caudal as dorsal raphe nucleus and the substantia nigra, which is ≥5 mm away from graft core (Figures 2G and 2I). At 6 months post-transplantation we confirmed that all starter cells (as detected by GFP expression) co-expressed human nuclei (HuNu; Figure 2H). At this time point, traced mCherry+/GFP− host neurons could be found in the striatum, close to the graft core, as well as in distal structures matching those observed at 6 weeks (Figure 2J). Quantifications showed that the majority of neurons connecting to the graft at this time point were located in the cortex, but connected neurons were also detected in thalamus, striatum, substantia nigra, and amygdala (Figure 2K; n = 5). The traced neurons in the prefrontal cortex were located mainly in layers III/V, displaying a classical pyramidal (Figure 3A) or basket cell (Figure 3B) morphology, and co-expressed the cortical marker TBR1 (Figure 3C). In the thalamus, the traced neurons were located in all anterior intralaminar nuclei (central medial, paracentral, and central lateral) (Figures 3D and 3F), which are the thalamic regions that normally project to the striatum (Van der Werf et al., 2002). These thalamic cells expressed Calbindin (CALB), but not Parvalbumin (PV) (Figures 3G and 3H). The traced host striatal cells connecting the grafted cells were distributed around the periphery of the graft and displayed a typical MSN morphology (Figures 2D″ and 3I) and expressed DARPP32 (Figure 3I). Thus, the connectivity observed after 6 weeks remained stable for up to 6 months post-transplantation, and the host brain regions that provide input to the grafted human neurons matched those that connected to endogenous striatal neurons. In the next set of experiments, we altered our experimental design to trace graft-to-host connectivity. Again, we used 6-OHDA lesioned nude rats, but this time the rats had been pre-injected with the tracing or control lentiviral vectors 4 weeks prior to transplantation. These virus injections were placed into two regions known to be densely innervated by transplanted human DA neurons: striatum (n = 6 for tracing and n = 4 for control) and prefrontal cortex (n = 5 for tracing and n = 4 for control). Thus, on the day of transplantation, the host brain contained GFP+ TVA-GP-expressing starter neurons and served as the recipient environment for the transplanted midbrain patterned WT hESCs (Figures S3A and S3B). Six months after transplantation, cells in the grafts had matured into functional DA neurons as confirmed by recovery in amphetamine-induced rotation (Figure S3C). At this time point, ΔG-rabies was injected at the same host brain regions that were initially injected with lentiviral vectors, resulting in selective infection of the modified host neurons via the TVA receptor. Subsequently, retrograde labeling of transplanted hESC-derived neurons that connected to host neurons in these regions should occur in animals pre-injected with tracing, but not control, vectors.