Target-specific forebrain projections and appropriate synaptic inputs of hESC-derived dopamine neurons grafted to the midbrain of parkinsonian rats

Abstract

Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are a promising unlimited source of cells for cell replacement therapy in Parkinson's disease (PD). A number of studies have demonstrated functionality of DA neurons originating from hESCs when grafted to the striatum of rodent and non-human primate models of PD. However, several questions remain in regard to their axonal outgrowth potential and capacity to integrate into host circuitry. Here, ventral midbrain (VM) patterned hESC-derived progenitors were grafted into the midbrain of 6-hydroxydopamine-lesioned rats, and analyzed at 6, 18, and 24 weeks for a time-course evaluation of specificity and extent of graft-derived fiber outgrowth as well as potential for functional recovery. To investigate synaptic integration of the transplanted cells, we used rabies-based monosynaptic tracing to reveal the origin and extent of host presynaptic inputs to grafts at 6 weeks. The results reveal the capacity of grafted neurons to extend axonal projections toward appropriate forebrain target structures progressively over 24 weeks. The timing and extent of graft-derived dopaminergic fibers innervating the dorsolateral striatum matched reduction in amphetamine-induced rotational asymmetry in the animals where recovery could be observed. Monosynaptic tracing demonstrated that grafted cells integrate with host circuitry 6 weeks after transplantation, in a manner that is comparable with endogenous midbrain connectivity. Thus, we demonstrate that VM patterned hESC-derived progenitors grafted to midbrain have the capacity to extensively innervate appropriate forebrain targets, integrate into the host circuitry and that functional recovery can be achieved when grafting fetal or hESC-derived DA neurons to the midbrain.

Keywords: Parkinson's disease; RRID: AB_10807945; RRID: AB_11034569; RRID: AB_1141717; RRID: AB_177511; RRID: AB_2333092; RRID: AB_300798; RRID: AB_390204; RRID: AB_572263; RRID: AB_627128; cell transplantation; dopaminergic neurons; human embryonic stem cells; rabies-based tracing.

Figure 1

Temporal assessment of intranigral graft survival, differentiation, and axonal outgrowth. (a) Schematic overview of experimental time‐plan of the three experimental groups assessing fiber outgrowth at 6, 18, and 24 weeks post‐transplantation in a 6‐OHDA lesioned rat model. Location and size of the transplant was confirmed using immunohistochemistry for tyrosine hydroxylase (TH) at 6 weeks (b), 18 weeks (b₁), and 24 weeks (b₂). (c) Quantification of graft volume reveals no significant change in volume from 6 weeks (n = 6), 18 weeks (n = 6), and 24 weeks (n = 5). (d) 3D representation of graft fiber outgrowth, based on the extent of hNCAM immunohistochemistry at 6 weeks (d), 18 weeks (d₁), and 24 weeks (d₂). The needle represents the site of intranigral transplantation. Scale bars: (b, b₁, b₂) = 200 μm. Hip = hippocampus; SNc = substantia nigra pars compacta; SNr = substantia nigra pars reticulata; Str = striatum; TH = tyrosine hydroxylase; Tha = thalamus; Tx = transplant

Figure 2

Progressive reinnervation of host forebrain target structures by graft‐derived fibers. (a) 3D template of the host brain showing the position of the 12 anterior/posterior levels (white circles labeled 1–12) used to measure graft‐derived axonal outgrowth at 6, 18, and 24 weeks in (c). (b) Quantification of the percentage of host target structure volume innervated by hNCAM⁺ fibers at 6 weeks (n = 6), 18 weeks (n = 6), and 24 weeks (n = 5) post‐grafting, demonstrates the progressive ramification of hNCAM⁺ fibers over time. (c) Area innervated by hNCAM⁺ fibers at 12 selected anterior/posterior levels (as shown in (a)) at 6 weeks (n = 6), 18 weeks (n = 6), and 24 weeks (n = 5) shows continuous outgrowth and progressive reinnervation of forebrain target structures over time. Immunostaining for hNCAM (green) and NeuN (blue) in the striatum demonstrates that graft‐derived fibers are confined to white matter tracts at 6 weeks (white arrow in (d)), expanding into a fiber network in the striatal gray matter at 24 weeks (white arrowhead in (e)). (f) Quantification of hNCAM⁺ fiber density in whole striatum and nucleus accumbens at 6 weeks (n = 6), 18 weeks (n = 6), and 24 weeks (n = 5) confirms progressive innervation of dopamine target areas over time. Scale bars: (d, e) = 100 μm. cc = corpus callosum; GP = globus pallidus; Hip = hippocampus; Hyp = hypothalamus; NAc = nucleus accumbens; PFC = prefrontal cortex; Rap = raphe nucleus; SNc = substantia nigra pars compacta; SNr = substantia nigra pars reticulata; Sep = septum; Tha = thalamus; vStr = ventral striatum

Figure 3

Graft‐derived reinnervation of A9 and A10 target structures. (a–d) Analysis of graft‐derived innervation as revealed by hNCAM immunohistochemistry 6 weeks post‐transplantation, demonstrates no detectable graft fibers in PFC (b) and only few scattered axonal terminals in NAc (c) and dorsolateral striatum (d). (e–h) At 18 weeks, hNCAM⁺ fibers were observed in the PFC (f), with extensive graft‐derived innervation of the NAc (g). Individual hNCAM⁺ fibers can be detected in dorsolateral striatum (h) at this timepoint. (i–l) At 24 weeks, abundant hNCAM⁺ innervation was readily detectable in PFC (j) and NAc (k). At this timepoint, an extensive network of graft‐derived axonal projections were observed also in dorsolateral striatum (l). Black arrow in (b) and (j) denotes the lateral ventricle. Scale bars: (a, e, i) = 1 mm. (b–d, f–h, j–l) = 100 μm. Insets in (b–d, f–h, j–l) = 100 μm. ac = anterior commissure; DL = dorsolateral; fmi = forceps minor; LV = lateral ventricle; NAc = nucleus accumbens; PFC = prefrontal cortex; Sep = septum; Str = striatum

Figure 4

Outgrowth of graft‐derived dopaminergic fibers and related functional outcome. (a–c) Analysis of double positive hNCAM⁺/TH⁺ fibers at different levels in the host brain at 6 weeks post‐grafting revealed the presence of some graft‐derived dopaminergic fibers coursing through the cerebral peduncle/nigrostriatal pathway (a), reaching the caudal portion of the striatum (b). Despite the presence of lesion‐spared endogenous dopaminergic fibers (hNCAM⁻/TH⁺), no double positive fibers were detected in dorsolateral striatum at this stage (c). (d–f) After 24 weeks, dopaminergic fibers of graft origin were seen coursing along the cerebral peduncle/nigrostriatal pathway in large numbers (d), providing dense innervation of the caudal striatum (e). Significant hNCAM⁺/TH⁺ innervation was readily detectable in dorsolateral striatum at this stage (f). (g–i) Analysis of graft‐derived dopaminergic innervation in one animal in the 24‐week group with no behavioral recovery showed few hNCAM⁺/TH⁺ fibers coursing via the cerebral peduncle/nigrostriatal pathway (g), with scattered fibers reaching the caudal striatum (h). Sparse double positive fibers can be seen reaching dorsolateral striatum (i) in this animal. (j) Quantification of hNCAM⁺/TH⁺ positive fibers in dorsolateral striatum at 6 weeks (n = 5), 18 weeks (n = 5), and 24 weeks (n = 6) revealed a progressive increase in the density of graft‐derived dopaminergic fibers in this structure over time (Kruskal–Wallis, χ² ₍₂₎ = 7.658, p < .01, Dunn's multiple comparisons test revealed a significant difference between the 6 and 24 week groups, p < .05). (k) Assessment of amphetamine‐induced rotational bias pre‐ and post‐transplantation revealed progressive graft‐mediated functional recovery at 6 weeks (n = 4; squares in (k)), 18 weeks (n = 4; triangles in (k)), and 24 weeks (n = 3; circles in (k)). Animals that received grafts of human fetal midbrain tissue (n = 6; diamonds in (k)), demonstrated significant recovery (t ₅ = 4.743, p < .01) at 24 weeks post‐transplantation. (l) Analysis of graft‐derived dopaminergic innervation of the dorsolateral striatum by fetal tissue transplants reveals several hNCAM⁺/TH⁺ fibers (white arrow). Scale bars: (a, d, g) = 100 μm. (b, c, e, f, h, i) = 100 μm. (l) = 20 μm. * = p < .05; ** = p < .01; CP/NSP = cerebral peduncle/nigrostriatal pathway; DL = dorsolateral

Figure 5

Synaptic host‐to‐graft integration revealed by monosynaptic tracing at 6 weeks matches endogenous connectivity. (a) Experimental design for assessment of endogenous connectivity using monosynaptic tracing. LV‐helper was used to generate starter neurons in midbrain and mCherry expressing EnvA‐pseudotyped ΔG‐rabies was used as a retrograde transsynaptic tracer to map inputs to midbrain. (b) 3D representation of endogenous synaptic inputs to midbrain of normal un‐lesioned rats, as revealed by rabies tracing. Each dot represents a traced neuron (mCherry⁺) in a 1:8 series of mCherry immunostained sections from a representative animal. (c) 3D representation of whole brain monosynaptic inputs to grafted neurons at 6 weeks revealed a pattern of traced neurons similar to that obtained from the mapping of endogenous inputs to the SN seen in (b). Histological analysis of transplants after 6 weeks revealed that a large proportion of grafted neurons expressed GFP and were selectively infected by mCherry expressing rabies (d). A significant proportion of starter neurons (GFP⁺/mCherry⁺) also expressed TH (e, e₁, e₂). (f) Heat map representing the percentage of mCherry⁺ labeling in each defined host brain area, divided by the total number of traced neurons per brain. Color grades represent different density of labeling, with associated values (3–18%) expressed in the legend. Data compiled from six individual animals (n = 6). Analysis of traced neurons (mCherry⁺) in the host brain revealed extensive synaptic inputs to the grafted neurons originating from structures such as somatosensory cortex (g), striatum (h), hypothalamus (i), and dorsal raphe nucleus (j). Scale bars: (d) = 100 μm. (e, e₁, e₂) = 40 μm. (g–j) = 200 μm. Amg = amygdala; Caud str = caudal striatum; Ctx = cortex; DL str = dorsolateral striatum; GP = globus pallidus; Hyp = hypothalamus; M ctx = motor cortex; PFC = prefrontal cortex; PVH = paraventricular hypothalamic nucleus; RN = raphe nucleus; SColl = superior colliculus; SN = substantia nigra; Som ctx = somatosensory cortex; Str = striatum; Tha = thalamus; VM str = ventromedial striatum; VTA = ventral tegmental area

Figure 6

Identification of the phenotype of traced host neurons. Immunostaining for specific phenotypic markers reveals the identity of host inputs to graft. Labeled neurons in striatum co‐expressed μ‐opiod receptor (MOR) (a, a₁), neurons in the subthalamic nucleus (STN) co‐expressed Barhl1 (b, b₁), while those in the dorsal raphe nucleus (RN) co‐expressed serotonin (5‐HT) (c, c₁). White arrow denotes double positive neurons for mCherry and phenotypic marker. Scale bars: (a–c₁) = 20 μm. STN = subthalamic nucleus; RN = rape nucleus; 5‐HT = serotonin