Human neural stem cell line (STROC05)
conditionally immortalized human striatal neural stem cell line (STROC05, kindly provided by ReNeuron Ltd., Surrey, UK) was previously described
]. In brief, STROC05 cells were isolated from the whole ganglionic eminence of 12-weeks-old human fetal brain. The cmyc-ERTAM
gene was transfected into cells with the retroviral vector pLNCX-2 (Clontech). Transfected cell colonies were isolated following neomycin selection before being expanded into a clonal cell line
]. To maintain proliferation through the conditional immortalization gene, 4-hydroxy-tamoxifen (4-OHT, 100 nM/ml; Sigma-Aldrich, UK) was added to proliferation media. The STROC05 cell line was expanded in T75 tissue culture flasks (Falcon, UK). Flasks were coated with mouse laminin at a concentration of 1:100 (mouse, 10 μg/ml; Trevigen, USA) for at least 2 hours at 37°C. Medium was changed every 2 days and cells were passaged at 90% confluence. The expansion media consisted of Dulbecco’s Modified Eagle’s Medium/Ham’s F12 (DMEM:F12; Gibco, UK) which was supplemented with additional components (Table
). To stimulate proliferation, growth factors, such as basic fibroblast growth factor-2 (bFGF-2, 10 ng/ml; Peprotech, UK) and epidermal growth factor (EGF, 20 ng/ml; Peprotech, UK), were added to the media.
Composition of cell culture media to expand the STROC05 cell line
In vitro differentiation of STROC05 cells
To induce neuronal differentiation and increase the proportion of DARPP-32 cells, STROC05 cells were grown in vitro
for 21 days on laminin (mouse, 10 μg/ml, Trevigen) and poly-l-lysine (PLL, 100 μg/ml, Sigma) coated T175 flasks with 90% confluence, as previously described
]. For the first week, differentiation was induced using media that contained all components from the proliferation media, with the exception of bFGF-2, EGF and 6-OHT. For the 2nd
week of differentiation, media consisted of neurobasal media (Gibco) supplemented with B-27 (Gibco), L-Glutamate (Sigma) and Purmorphamine. For the 2nd
week of differentiation, bFGF was added again to the media as a survival factor
] and to promote a rostral positional specification of neurons
], but was omitted again for the 3rd
week of differentiation as positional specification in most cells is completed. Purmorphamine (1 μM, Calbiochem) was added to the culture media throughout the 3 weeks of differentiation.
Effect of harvesting on cell viability and differentiation
As differentiated cells are very vulnerable when removed from tissue culture flasks, it is essential to establish whether harvesting these cells after long-term differentiation affects their viability and differentiation status. For this, cells were harvested with Trypzean EDTA for less than five minutes at 37°C, followed by adding a soybean trypsin inhibitor to inactivate the enzymatic activity. After harvesting, cells were centrifuged for 5 minutes at 1500 rpm and the cell pellet was re-suspended in 1 ml of DMEM. Using the trypan blue exclusion test, cells were counted and viability was established to be 89.5%. Cells were re-seeded on laminin-coated cover slips in 24 well plates at 100,000 cells per well. After 24 h, viability of these re-seeded conditions was evaluated again using the live/dead stain (viability/cytotoxicity kit for mammalian cells, Gibco) and compared to cells that were not harvested. For the live/dead stain, media was aspirated and cells were washed once with PBS prior to incubation with 2 μM calceinAM (to detect live cells) and 4 μM ethidium homodimer-1 (EthD-1) (to detect dead cells) in PBS (500 μL per well) for 45 minutes at 37°C. Photos were taken immediately using a fluorescent microscope (Zeiss). A separate set of coverslips were fixed with 4% Parafix (Pioneer) for 5 min. Immunohistochemistry was used to establish if harvesting of cells would affect the proportion of neurons (1:500, mouse anti-β-III-tubulin, Tuj, AB7751, Abcam) and specifically DARPP-32 neurons (1:500, rabbit anti-DARPP-32, AB1656, Chemicon) within the cell suspension. After overnight incubation (at room temperature) with the primary antibody, an appropriate secondary ALEXA594 (1:1000, Molecular Probes) was applied for 60 min prior to attaching the coverslips to microscopic slides with Vectashield for fluorescence containing DAPI (Vector Laboratories). Total DAPI, as well as Tuj and DARPP-32 cells, were counted under a Zeiss Axioscope.
All procedures of this study were carried out according to the UK Animals (Scientific procedures) Act 1986 (PPL70/6445), as well as the ethical review process of King’s College London. A widely used and well characterized mouse transgenic model of Huntington’s disease, R6/2 mice present with a rapid disease onset that is evident as early as 6 weeks of age. Especially the development of a clear behavioural phenotype in the R6/2 compared to the N171-82Q or HDH111 is important to establish a potential therapeutic efficacy.
The average life span of R6/2 mice with 210 CAG repeats is approximately 16 weeks of age
]. Here, R6/2 mice were generated from a colony that was maintained by backcrossing R6/2 males to (CBA × C57BL/6) F1 females (B6CBAF1/OlaHsd, Harlan, UK). Mice were kept in standard housing conditions, on a standard chow diet with water available ad libitum
. During the last 2 weeks of the study (12 and 13 weeks of age), a mash diet was prepared by soaking chow pellets in water. These were placed in the floor of the cages within easy reach of the motor impaired R6/2 mice. Transgenic mice were identified by Polymerase Chain Reaction (PCR) on an ear tissue sample at 4 weeks of age, as previously described
Forty female mice were randomized into 4 groups; wild type mice receiving vehicle (WT-veh, n
10), R6/2 mice receiving vehicle (R6/2-veh, n
10), R6/2 mice receiving undifferentiated cells (R6/2-undiff, n
10), and R6/2 mice receiving long-term purmorphamine-differentiated cells STROC05 (R6/2-diff, n
10). Mice were group-housed 4 per cage containing mixed genotypes (one from each experimental group) to ensure comparable standard housing conditions, as described by Hockley et al.
On the day of transplantation, the cells were harvested by incubation with Trypzean EDTA for less than five minutes at 37°C, followed by adding soybean trypsin inhibitor to inactivate the enzymatic activity. After harvesting, cells were centrifuged for 5 minutes at 1500 rpm and the cell pellet was re-suspended in 2 ml of DMEM for cell counting. Cells were suspended in vehicle consisting of 2.5 ml of DMEM and 3.75 ml of Hypothermosol (BioLife Solutions) at a concentration of 7.5 × 104 cells/μl. Using the trypan blue exclusion test, viability was determined to be 89%.
At 7 weeks of age, mice underwent stereotactic surgery for the injection of NSCs. This allowed sufficient time to conduct pre-implantation MRI scans, as well as behavioural test, after animals were weaned at 4 weeks of age from their mothers. Additionally, animals’ genotype was determined and animals were randomly allocated to their experimental groups based on a sequence of random numbers. Although at 7 weeks of age, R6/2 mice do not exhibit a motor deficit
], they do nevertheless already show signs of brain atrophy
]. Impor-tantly, R6/2 mice do not display any neuronal loss
]. At this age, there is also a decrease, as well as morphological abnormalities, in microglia
For cell implantation, anaesthesia was induced through isoflurane inhalation (Abbott) at 4-5%, then maintained at 1.5-2%. Animals were mounted in a stereotaxic frame and a sagittal incision was carefully made followed by the drilling of two burr holes. Either 6 μl (3 μl per side, 0.5 μl/min) of cells or vehicle were injected with a 22 Gauge needle attached to 10 μl Hamilton syringe using a convection-enhanced delivery
] at Anterior-Posterior +0.5 mm (in relation to Bregma), Lateral ±2 mm and −3.5 mm below the surface of the dura. The deposit was divided into two equal amounts; one was injected at −3 mm (after retraction of the needle by 0.5 mm) and the other at −2.5 mm. After injection, the syringe was left in place for 5 minutes and slowly withdrawn over 3 minutes, followed by suturing of the incision. During the surgery, body temperature was controlled using a homeostatic heating pad set at 37°C. No immunosuppression was given as STROC05 cells exhibit a robust survival in the 3-nitropropionic acid rat model of Huntington’s disease over 90 days (Additional file
: Figure S1), as well as wild-type mice (Additional file
: Figure S2).
After surgery, post-operative care included fluid-replacement (0.1 ml saline/animal) and a local analgesic (EMLA cream 5%; AstraZeneca, UK). The animals were singly caged with softened food pellets and water available ad libitum for 24 h before being returned to their home cages.
Weight loss is a prominent symptom in R6/2 mice
]. Body weight has often been used as a reliable outcome measure to assess the beneficial effect of different therapeutic approaches in R6/2 mice
]. Mice were weighted weekly from the time of weaning (4 weeks) until the end of the study. To avoid the impact of diurnal feeding habits, body weight was obtained weekly on the same day and time.
For each behavioural test, the running order of animals was based on a randomization of the cages, but within each cage (containing WT and R6/2), mice were run sequentially. Animals within each cage were randomly chosen for each trial. If more than one trial was conducted, this was run in the same sequence.
The rotarod is considered a very sensitive and reliable motor task to assess motor coordination in HD transgenic mice
]. R6/2 mice are known to have impaired rotarod performance
]. According to a standard protocol
], mice were placed on a rotarod (Ugo Basile) with a 3 cm diameter rod at a constant speed of 4 rpm for 20 sec. After this acclimatisation period, the rod speed accelerated from 4 to 40 rpm over 300 sec. Latency for mice to fall from the rod was recorded. Rotarod performance was assessed over three successive days with 3 trials per day. The first assessment day was always excluded from analysis. Mice were tested one week pre-transplantation, as well as at 1, 3, and 5 weeks post-transplantation.
The open field test has been used extensively as a reliable measure to evaluate locomotor activity and anxiety-like behaviour in R6/2 mice
]. A custom-built 100 cm diameter and 35 cm deep circular open field arena (Engineering & Design Plastics) was divided into outer and inner zones by a circle drawn 4 cm from the outer walls. Mice were placed individually in the outer zone facing the centre of the maze with their behaviour being automatically recorded by a camera for a period of 5 min. Data was subsequently analysed using Ethovision XT7.0 software (Noldus). The arena was cleaned between mice to prevent behavioural influences from the odours of previous trials. Total distance travelled (locomotion) and time spent in the outer zone (thigmotaxis, indicative of anxiety-like behaviour) were measured one week pre-transplantation, as well as at 1, 3, and 5 weeks post-transplantation.
Grip strength analysis is a reliable and sensitive test to evaluate muscular strength in R6/2 mice
]. To measure forelimb grip strength, mice were lowered towards the grid to grab it with both front paws. Mice were gently pulled back until they released their grip and the equipment automatically measured the force required to pry the mouse from the grid. A single session consisting of 5 consecutive trials was recorded once a week at 4, 5, and 6 weeks post-grafting. As low scores may be due to the mouse failing to grip the grid effectively, the best three scores of the five trials were averaged.
Magnetic Resonance Imaging (MRI)
Six weeks following cell implantation, mice were anesthetised using isoflurane (4-5% induction, 1.5-2% maintenance in 0.7 l/min medical air and 0.3 l/min oxygen) and fixed within a head holder/respiration mask to reduce head movement. MR images were acquired using a 7 Tesla magnet (Varian), equipped with a 100 Gauss gradient set and a 39 mm transmission/receive coil (Rapid). A T2
-weighted multi-echo multi-slice (MEMS) sequence was used (TR
2500 ms, minimum TE
10 ms, number of echo
8, echo spacing
10 ms, averages
128x128, and FOV
20 × 20 mm). Thirty coronal slices with 0.5 mm thickness were acquired across the mouse brain. Manual segmentation of anatomical regions of interest (ROIs, Additional file
: Figure S3), including whole brain, striatum, cortex, hippocampus, and lateral ventricle, was performed using JIM 5.0 (Xinapse). Criteria used to define ROIs are summarized in Table
. Manual segmentation of the same structure at two separate occasions yielded an intra-rater discrepancy of less than 2% error.
Anatomical criteria adopted to manually segment ROIs
After MRI scanning, anesthetized animals received an intracardial perfusion of saline followed by 4% Parafix (Pioneer). Brains were excised and post-fixed for 24 h at 6°C before being cryoprotected in 30% sucrose at 6°C. Sections (40 μm) were cut on a freezing sliding microtome in the coronal plane and stored at −20°C in tissue cryoprotective solution (25% glycerine, 30% ethylene glycol, and 50% PBS).
To identify transplanted cells, sections were stained with a mouse anti-human nuclear protein (HNA) antibody (1:400, MAB1218, Millipore). For this, sections were rinsed with PBS, blocked for 30 minutes in 0.1%
as inhibitor for endogenous peroxidase activity (Sigma), followed by 60 min incubation in 10% blocking solution (10% normal goat serum in 0.3% Triton X-100 PBS) at room temperature (RT, 21°C). To block the non-specific binding of endogenous biotin, the sections were incubated with avidin-biotin blocking solutions (Vector) for 30 min. The sections were incubated with the HNA antibody at RT for an hour, followed by 10 min of incubation at RT with secondary biotinylated anti-mouse antibody (1:200, Vector), and 5 min at RT with an avidin-biotinylated-peroxidase complex (1:100 in PBS, Vector). Secondary antibody binding was visualized using 3,3’-diaminobenzoic acid (DAB, Sigma) dissolved in PBS with the addition of H2
to a concentration of 0.03% immediately before use. Finally, the sections were washed in PBS, mounted onto glass slides, dehydrated for 5 min in each of 70, 85, 90, and 100% alcohol, cleared by xylene, and coverslipped with Entellen (Merck, UK).
For fluorescence immunohistochemistry, sections were incubated for 60 min in 10% blocking solution (10% normal goat serum in 0.3% Triton X-100 in PBS) at RT, followed by 30 min of an avidin-biotin blocking solution (Vector). Sections were then incubated with appropriate primary antibodies against transplanted cells (mouse anti-HNA 1:400), neurons (rabbit anti-Fox3, 1:500, ab104225, Abcam), or DARPP-32 neurons (rabbit anti-DARPP-32, 1:500). After overnight incubation with the primaries, an appropriate secondary antibody (1:200, ALEXA 350; 1:500, ALEXA 647, Molecular Probes) was applied for 60 minutes at RT. Sections were rinsed in PBS and mounted in Vectashield with DAPI.
Statistical analyses were conducted using GraphPad Prism 5.03 (GraphPad Software, San Diego, California, USA) to determine significant differences (p
.05) between in vitro
and post-mortem immunohistochemistry (independent samples t
-test), as well in vivo
measures (repeated-measures two-way ANOVA). Bonferroni post-hoc tests were applied if ANOVAs revealed a significant result. All error bars on graphs are displayed as the standard error of the mean (SEM).