Supplementary Fig. 1. Illustration and rating scale of the beam walking test. Mice were trained to cross an elevated round beam with 9 mm of diameter, 40 cm length and raised at 21 cm height, to reach the escape platform. The latency to cross the beam (up to 60 sec) and the motor coordination applied during the test was recorded. Motor coordination was evaluated by the way mice crossed the beam, (A) correct crossing, (B) upside down crossing (wrong crossing), and half correct crossing, and scored according to a pre-defined rating scale (C). The performance was scored from 0 to 180. *1- time expended to cross the beam; *2- the animal crossed the beam correctly; *3- the animal crossed at least 20 cm of the beam correctly and the remaining beam upside down; *4- the animal crossed all the beam upside down.
Supplementary Fig. 2.cerebellar NSC and svzNSC have the same potential to give rise to neural cells and to originate mature functional neurons upon differentiation. Immunocytochemistry fluorescence images revealed that (A-C) cerebellar NSC and (D-F) svzNSC upon differentiation originated mixed cultures composed by astrocytes shown by GFAP (green), neurons shown by β3tubulin (red) and oligodendrocytes (not shown); DAPI (blue). (G-H) The variations of intracellular concentrations of cerebellar NSC (CRB) and svzNSC (SVZ) differentiated cultures, through single cell calcium imaging analysis, demonstrated that there is no significant difference at the ability to originate (G) mature functional neurons and (H) progenitors cells between cerebellar NSC and svzNSC. Mature functional neurons respond to KCl stimulus (neuronal-like response) whereas, cells that remain in "stem cell state" respond to histamine stimulation (progenitors-like response). Panel C is repeated in Fig. 1.B. Data are presented as mean ± SEM; n=3; p > 0.05; unpaired Student´s t test.
Supplementary Fig. 3.The large majority of cerebellar NSC 8 weeks after cerebellar transplantation are not apoptotic.Cerebellar sections of mice injected with cerebellar NSC expressing GFP were assessed for apoptotic cell death. (A-C) Widefield fluorescence microscopy images, (D) confocal fluorescence images and (E-G) 3D reconstructions obtained from the confocal z-stack of cerebellar sections, assessed for apoptotic cell death with TUNEL assay (red), reveal that the majority of the transplanted cerebellar NSCare not in apoptosis. The white arrows indicate cells positive for TUNEL assay. (H-J) Immunohistochemicalwidefield fluorescence microscopy images of mice´s cerebellar sections reveal that the majority of the transplanted cells are negative for the apoptosis marker cleaved caspase-3. Scale bar of lower panels = 20 µm.
Supplementary Fig. 4. Upon transplantation into mice´s cerebellum cerebellar NSC differentiate into neurons, astrocytes and oligodendrocytes. Immunohistochemical 3D reconstruction of confocal z-stack fluorescence images of mice´s cerebellar sections 8 weeks after the transplantation of cerebellar NSC expressing GFP (green). (A) Some cerebellar NSC remain undifferentiated expressing SOX2 (red). White arrows indicate undifferentiated cerebellar NSC expressing both GFP and SOX2 and the yellow arrow indicate the zoom region of GFP positive/SOX2 positive cells in the lower panel. While other cerebellar NSC differentiate into (A) SOX-2 negative cells (blue arrows), (B) neurons shown by β3tubulin (red), (C) astrocytes shown by GFAP (red) and (D) oligodendrocytes shown by NG2 (red) (blue arrows).
Supplementary Fig. 5.cerebellar NSC did not differentiate into new Purkinje cells. Immunocytochemistry fluorescence images revealed (A and C) no Purkinje cells, shown by calbindin (green), upon cerebellar NSC differentiation during 14 days. Whereas, neurons shown by β3tubulin (red) were detected. Fluorescence microscopy images acquired with a 40x objective on a ZeissAxiovert 200 imaging microscope.
Supplementary Fig. 6. Mutant ATXN3 mRNA levels are not reduced by cerebellar NSC transplantation. The mRNA relative levels of the cerebellum of Machado-Joseph disease transgenic mice injected with cerebellar NSC or with HBSS were assessed 4 weeks after the injection by qRT-PCR. No significant difference was observed between the mutant ATXN3 mRNA levels of the mice injected with cerebellar NSC (mutant ATXN3 NSC) and the mice injected with HBSS (mutant ATXN3 HBSS). NSC-transplanted mice (n=11) and HBSS-injected mice (n=8). The quantification was performed with the Paffl method and usingHPRT as housekeeping gene. Data are presented as mean ± SEM; p > 0.05; unpaired Student´s t test.
Supplementary Fig.7.cerebellar NSC transplantation reduces the pro-inflammatory mediators and increases neurotrophic factors levels and the glutamatergic receptors expression. ThemRNA relative levels of the cerebellum of Machado-Joseph disease transgenic mice non-injected (Tg), injected with cerebellar NSC (Tg NSC) or with HBSS (Tg HBSS) or wild type mice (Wt) were assessed 4 weeks after the injection by qRT-PCR. (A) The neurotrophic factors BDNF and NGF were evaluated. The BDNF expression on the mice injected with cerebellar NSC (BDNF Tg NSC) as compared to the mice injected with HBSS (BDNF Tg HBSS) and with wild type mice was increased. (B) In the pro-inflammatory mediators evaluated, IL1β and IL6, a significant decrease was observed in the IL1β levels of the mice injected with cerebellar NSC (IL1β Tg NSC). Transgenic mice have higher levels of these interleukins as compared to wild type mice. Moreover, the IL1β levels of transgenic mice injected with HBSS (IL1β Tg HBSS) are decreased as compared to non-injected transgenic (IL1β Tg). (C) GABA A receptor subunit beta 2 (GABAR) and glutamate receptor 3 (GluR3) were also evaluated and a significant increase of the GluR3 after cerebellar NSC transplantation was detected. Wild type mice have higher levels of these neuronal receptors as compared to transgenic mice. NSC-transplanted mice (Tg NSC, n=11), HBSS-injected mice (Tg HBSS, n=8), non-injected transgenic mice (Tg, n=5). The quantification was performed with the Paffl method and using HPRT as housekeeping gene. Data are presented as mean ± SEM. ***p < 0.001, **p<0.01 and *p <0.05 when comparison was established between the conditions indicated by the lines; ##p<0.01 and #p<0.05 when comparison was established with Tg HBSS mice.One-way ANOVA analysis of variance combined with Bonferroni post-test were used for multiple comparisons.
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