Chemical Control of Grafted Human PSC-Derived Neurons in a Mouse Model of Parkinson's Disease

Abstract

Transplantation of human pluripotent stem cell (hPSC)-derived neurons is a promising avenue for treating disorders including Parkinson's disease (PD). Precise control over engrafted cell activity is highly desired, as cells do not always integrate properly into host circuitry and can cause suboptimal graft function or undesired outcomes. Here, we show tunable rescue of motor function in a mouse model of PD, following transplantation of human midbrain dopaminergic (mDA) neurons differentiated from hPSCs engineered to express DREADDs (designer receptors exclusively activated by designer drug). Administering clozapine-N-oxide (CNO) enabled precise DREADD-dependent stimulation or inhibition of engrafted neurons, revealing D1 receptor-dependent regulation of host neuronal circuitry by engrafted cells. Transplanted cells rescued motor defects, which could be reversed or enhanced by CNO-based control of graft function, and activating engrafted cells drives behavioral changes in transplanted mice. These results highlight the ability to exogenously and noninvasively control and refine therapeutic outcomes following cell transplantation.

Figure 1. Establishment, mDA Neuron Differentiation, and Functional Assessment of the DREADD-Expressing hESC Lines

(A) Schematic diagram showing the strategy for functional regulation of human mDA neurons transplanted into PD mice. (B) Schematic depiction of the strategy for knockin of EGFP-, hM3Dq-, or hM4Di-expressing cassette into AAVS1 locus. Exons are shown as orange boxes. The vertical arrows indicate targeting site by sgRNA T2 in AAVS1 locus. Donor plasmids are as follows: SA-Pur, splice acceptor sequence followed by a T2A self-cleaving peptide sequence and the puromycin resistance gene, and CAG, synthetic CAGGS promoter containing the actin enhancer and the cytomegalovirus early promoter. mCherry is fused to the C terminus of receptors. (C) Immunostaining of day 42 cultures derived from hM4Di- or hM3Dq-expressing hESCs shows markers for mDA neurons. Ho, Hoechst. Scale bar, 25 µm. (D) Quantification of cellular differentiation presented in (C). (E and F) Whole-cell patch clamping of differentiated human mDA neurons shows representative changes in membrane potential on 10-week-old cultures (E) and spontaneous action potentials (sAP) on 12-week-old cultures (F) upon treatment with and wash of CNO (40 µM). (G and H) The statistical analysis of changes in membrane potential (G; n = 4–9 for each group) and frequency of spontaneous action potentials (H) upon treatment with and wash of CNO (40 µM) (n = 6–9 for each group). Data are represented as mean ± SEM. Paired t test. **p < 0.01, ***p < 0.001. See also Figure S1.

Figure 2. Maturation of and Innervation by Human DREADD-Expressing mDA Neurons in PD Mice

(A) TH expression and graft morphology at 6 months after striatal transplantation of human mDA neuron progenitors expressing hM4Di. Scale bar, 500 µm. Boxed area indicates graft location. Stereological analysis shows the total number of TH-positive cells in the graft expressing EGFP, hM4Di, or hM3Dq at 6months after transplantation. Data are presented as mean ± SEM; n = 8–10 for each group. (B) Immunostaining images show that the grafted TH-positive cells coexpress human nuclei (hN), FOXA2, and mCherry. Boxed regions are magnified on the right. Scale bar, 50 µm for large images, 10 µm for magnified images. (C) Immunostaining of hNCAM shows fibers from hM3Dq-expressing mDA neuron-enriched human graft 6 months after intrastriatal transplantation. Boxed areas are amplified below. White asterisk indicates the graft site. Scale bar, 500 µm. CPu, caudate-putamen; Tu, olfactory tubercle; NAc, nucleus accumbens; AC, anterior commissure. (D) Immunostaining shows outgrowth of fibers from hM3Dq graft and presence of fibers in the host tissue. Black asterisk indicates graft. Boxed area is amplified on the right. Scale bar, 25 µM. See also Figures S2–S4.

Figure 3. sEPSCs in Host GABA Neurons in Response to Activation of Human mDA Neuron-Enriched Grafts

(A, C, E, and G) Typical whole-cell patch-clamp recordings of sEPSCs in striatal GABA neurons of the brain slices that received EGFP (A) or hM3Dq (C, E, and G) cell transplantation with the absence or presence of CNO (40 µM) (A, C, E, and G), or presence of SCH-23390 (10 µM) (E) or LE300 (10 nM) (G). Holding potential is −70 mV. (B, D, F, and H) Cumulative distributions of the interevent intervals of sEPSCs for (A), (C), (E), and (G), respectively. **p < 0.01 in (D), K-S test. The insets show the mean sEPSC frequency before and after CNO treatment in the absence (B, n = 6; D, n = 8), or presence of 10 mM SCH-23390 (F, n = 4) or 10 nM LE300 (H, n = 5). Data are presented as mean ± SEM. Paired t test. ***p < 0.001.

Figure 4. Control of Motor Behaviors of Transplanted PD Mice

(A) Schematic depiction of the experimental process of animal model, transplantation, and behavioral analysis. s-Rotation, spontaneous rotation. (B) Amphetamine-induced rotation shows the changes of rotation behavior from all of the three groups grafted with mDA neurons expressing EGFP, hM4Di, or hM3Dq over the 5 months posttransplantation. n = 10 for each group. (C) Cylinder test shows the preferential ipsilateral touches before and after striatal transplantation of human mDA neurons as well as treatment with and 2 days after withdrawal of CNO. n = 10 for each group. (D) Rotarod test shows the changes in latency to fall before and after striatal transplantation of human mDA neurons as well as treatment with and 2 days after withdrawal of CNO. n = 10 for each group. (E and F) Spontaneous rotation test shows CNO-induced change in net ipsilateral rotations (E) and preferential ipsilateral rotations (F) in EGFP, hM4Di, and hM3Dq groups. n = 7–10 for each group. (G) CNO-induced preferential contralateral rotation was inhibited by combinatorial pretreatment of DA receptor antagonists (DRi) SCH-23390 and Raclopride. n = 10. Data are presented as mean ± SEM. One-way ANOVA followed by Tukey’s posthoc test in (C), (D), and (G). Paired t test in (E) and (F). *p < 0.05, **p < 0.01, ***p < 0.001. See also Movies S1, S2, S3, and S4.