Fig 2: Deficits in skilled reaching behavior in DARPP-32 Thr34A mutant mice.Endpoint measurements of reaching behavior are presented. (A) Success on the first reach attempt, in percentage. (B) Total success, in percentage. (A' and B') Bar graphs show the learning rate for each endpoint. The results are presented as means± SEM; n = 8 per group. Asterisks denote where DARPP-32 Thr34A mutant mice differ significantly (* P < 0.05, ** P < 0.01, *** P < 0.001) from wild type (WT) mice.

Fig 3: Development of HD-associated behavioural phenotypes following intraventricular injection of exosomes isolated from HD patient-derived fibroblasts and evidence of mHtt propagation within the brain. a Timeline of in vivo experimentation using exosomes isolated from HD143F. b Representative images of the simple swim test. c Plotting of the time to reach the platform as well as the latency to fall, as measured by the rotarod test, revealed cognitive and motor-related deficits in mice transplanted with exosomes carrying mHtt in comparison to mice injected with either exosomes derived from normal human embryonic fibroblasts or in sham animals. d Triple immunoflurorescent staining was further carried out for DARPP-32, EM48 and DAPI and revealed the propagation of mHtt from implanted exosomes isolated from HD patient-derived fibroblasts into the host striatum and striatal cells as early as 3 weeks post-implantation. Values are expressed as mean ± SEM. Statistical analyses were performed using the Student’s t test. *p < 0.05 and **p < 0.005 compared to hEF group. Numbers of mice used are as follows: hEF-exosome, n = 11; HD143F-exosome, n = 11; Sham, n = 9. Scale bar d = 20 µm. DARPP-32 dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa; DAPI 4',6-diamidino-2-phenylindole; HD143F-exo exosomes isolated from fibroblasts derived from an HD patient with 143 CAG repeats; hEF-exo exosomes isolated from human embryonic fibroblasts; LV lateral ventricle; P2 post-natal day 2; STR striatum; w weeks

Fig 4: Development of an immune/inflammatory response following striatal neuronal loss in mice transplanted with HD patient-derived fibroblasts. a Schematic representation of the immune/inflammatory response gradient within the striatum illustrating that the strongest reaction is in the vicinity of the injection site. b The bar graph shows increased astroglial and microglial responses in the striatum of mice implanted with HD143F compared to those implanted with hEF at 30 and 40 weeks post-injection (n = 3 mice per group). c Representative images of astroglial (GFAP; green) and microglial (ED1; red) responses 30 and 40 weeks following the engraftment of fibroblasts from HD143F or hEF cells. Values are expressed as mean ± SEM. Statistical analyses were performed using the Student’s t test. *p < 0.05; **p < 0.005 compared to hEF group. The number of mice quantified in each group is indicated in each column of the graphs. d Representative images of the astroglial (GFAP; red) and microglial responses (ED1; red) surrounding neuronal (DARPP-32; green) and mHtt (EM48; red) staining. e Schematic summarizing the post-mortem observations following HD143F transplantation with respect to the corresponding timeline of analysis. Scale bars b = 30 µm; d = 50 µm. CTX cortex; DARPP-32 dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa; GFAP glial fibrillar acidic protein; HD143F fibroblasts derived from an HD patient with 143 CAG repeats; hEF human embryonic fibroblasts; LV lateral ventricle; STR striatum

Fig 5: Comparison of VM- versus FB-Patterned Graft CompositionPhenotypic characterization of hESC-derived VM-patterned (A–F) and FB-patterned cells (G–L) 6 months after grafting to the nigra.(A, B, G, and H) VM- (A) and FB-patterned cells (G) generated grafts of comparable size, as visualized by human NCAM staining. VM- (B) but not FB-patterned grafts (H) generated TH+ neurons in vivo.(C and I) In VM-patterned grafts (C) but not FB-patterned grafts (I), TH+ neurons also co-expressed the midbrain DA neuron marker FOXA2 (arrowheads).(D–F and J–L) FB- (J) but not VM-patterned graft cells (HuNu+) (D) expressed NKX2.1 and FOXG1 (E and K), and included DARPP-32+ neurons (F and L). See also Figure S1 for in vitro characterization of cell preparations.All graft neurons expressed a rabies tracing construct and, thus, nuclear GFP. Scale bars represent 1 mm (A, B, G, and H) and 20 µm (C–F and I–L). Images in (A), (B), (G), and (H) are digitally stitched from multiple high-magnification images. DARPP-32, dopamine- and cAMP-regulated phosphoprotein; FB, forebrain; FOXA2, forkhead box A2; FOXG1, forkhead box protein G1; hESCs, human embryonic stem cells; HuNu, human nucleus; NKX2.1, NK2 homeobox 1; TH, tyrosine hydroxylase; Tx, transplant; VM, ventral midbrain.