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Background Efforts in cell replacement therapy for Huntington’s disease (HD) are hindered by uncertainty of graft fate after transplantation. Current cell tracking methods fall short to either: 1) Detect labelled transplanted cells, or 2) Differentiate between live and dead cells, or 3) Provide long term tracking of growing grafts. Bioluminescence imaging (BLI) addresses these problems and could provide a cheaper, more reliable and less inconvenient alternative to currently used intracerebral cell tracking techniques. Aim We aimed to describe methods for long term tracking of transplanted Human Foetal Cells (HFC) using BLI in a HD rat model. Methods In-vitro: We have developed protocols to transduce Human Embryonic Kidney cells, Medium Spiny Neuron Progenitors and eventually HFCs with a lentivirus carrying a firefly luciferase gene. In-vivo: Using quinolinic acid lesioned adult female Sprague-Dawley rats as a HD model, 4 animals underwent transduced-cells transplantation followed immediately by BLI using intracerebral (IC) luciferin injection. These animals had pre and post-operative MRI for comparison. Five animals underwent BLI with IC luciferin at 12 weeks. Eight animals underwent acute BLI after transplantation using intraperitoneal (IP) Luciferin alternatives, namely: AkaLumin, D-Luciferin, D-Luciferin packaged in a nano vector and InfraLuciferin. Results In-vitro: All tested cell types were reliably transduced with a viral vector to express engineered luciferase. HFC viability inversely correlated with duration of incubation with the virus. In-vivo: HFC grafts were detected using BLI acutely and at 12 weeks after transplantation. D-Luciferin packaged in a nano vector showed promising results after IP injection, however signal is considerably weaker than IC injection. Conclusion BLI reliably and conveniently tracks transplanted HFC in small animals. This technique has great potentials in studying graft fate in terms of localization, viability, growth and integration.

Cell replacement therapies in Huntington’s disease (HD) aim to repair the nervous system by reintroducing the previously degenerated medium spiny neurons (MSNs) of the striatum and thereby restoring efferent and afferent projections. Despite significant advances in the generation of efficient protocols to differentiate pluripotent cells into authentic MSNs little is known about their functional efficacy. This study aimed to evaluate the survival, safety, integration, connectivity and functional efficacy of a hESC-derived cell therapy product (CTP) that was designed to be enriched for MSNs progenitors. Human CTP was stereotaxically transplanted into the striatum of a rat quinolinic acid model of HD. Rodents are treated with cyclosporine to prevent rejection and are being assessed on a range of behavioural tests sensitive to both motor and non-motor impairments over a period of 27 weeks to allow graft differentiation and development. While this is an on-going experiment, the following has been established with the current hESC-derived MSN CTP: (1) it consistently survives following transplantation into the rodent brain (as evidenced by bioluminescence imaging) and expresses markers of MSN differentiation (e.g., Darpp-32, CTIP2); (2) is safe (i.e., no evidence of neural overgrowth); (3) it is capable of forming functional synapses with the host brain and (4) and there is preliminary evidence of reduced motor bias in HD rats grafted with the hESC-derived CTP.

Primary human fetal cells have been used in clinical trials of cell replacement therapy for the treatment of neurodegenerative disorders such as Huntington's disease (HD). However, human fetal primary cells are scarce and difficult to work with and so a renewable source of cells is sought. Human fetal neural stem cells (hfNSCs) can be generated from human fetal tissue, but little is known about the differences between hfNSCs obtained from different developmental stages and brain areas. In the present work we characterized hfNSCs, grown as neurospheres, obtained from three developmental stages: 4–5, 6–7 and 8–9 weeks post conception (wpc) and four brain areas: forebrain, cortex, whole ganglionic eminence (WGE) and cerebellum. We observed that, as fetal brain development proceeds, the number of neural precursors is diminished and post-mitotic cells are increased. In turn, primary cells obtained from older embryos are more sensitive to the dissociation process, their viability is diminished and they present lower proliferation ratios compared to younger embryos. However, independently of the developmental stage of derivation proliferation ratios were very low in all cases. Improvements in the expansion rates were achieved by mechanical, instead of enzymatic, dissociation of neurospheres but not by changes in the seeding densities. Regardless of the developmental stage, neurosphere cultures presented large variability in the viability and proliferation rates during the initial 3–4 passages, but stabilized achieving significant expansion rates at passage 5 to 6. This was true also for all brain regions except cerebellar derived cultures that did not expand. Interestingly, the brain region of hfNSC derivation influences the expansion potential, being forebrain, cortex and WGE derived cells the most expandable compared to cerebellar. Short term expansion partially compromised the regional identity of cortical but not WGE cultures. Nevertheless, both expanded cultures were multipotent and kept the ability to differentiate to region specific mature neuronal phenotypes.

The transplantation of dopamine-rich tissue into the putamen of patients with Parkinson's disease shows much potential for use as a therapeutic strategy. However, a number of grafted individuals subsequently developed a set of abnormal involuntary movements (AIMs), unrelated to the dyskinesia caused by L-DOPA treatment, which have been termed graft-induced dyskinesia. Given the small number of patients, pre-clinical modeling of graft-induced dyskinesia in animal models will be critical to determine the underlying mechanisms and amelioration potential of this technique. Here we show that abnormal involuntary movements of the limbs, trunk and face can be observed in transplanted hemi-parkinsonian mice following amphetamine administration, similar to those previously described to model graft-induced dyskinesias in rats. C57Bl6 and CD1 mice were first rendered hemi-parkinsonian with 6-hydroxydopamine, treated with L-DOPA for 21 days until dyskinetic, and then transplanted with a single cell suspension of embryonic ventral mesencephalon (VM E12.5) tissue from corresponding strains into the denervated striatum. At 16 weeks post-transplantation, a single injection of amphetamine-elicited dyskinesia in a subgroup of mice of both strains, behavioural pattern not observed pre-transplantation. The number of surviving dopaminergic cells in the graft did not differ between those that developed AIMs and those that did not. The movements were phenotypically comparable to those seen in the rat model and parallels can be drawn to the human form of the movements, although the mouse model maybe less reproducible than the rat equivalent. This mouse model will facilitate assessment of graft-induced dyskinesia with mouse-derived stem cell lines and exploration of mechanisms using transgenic mice in future studies.

Background Cell replacement therapy (CRT) is a potential therapeutic intervention for Huntington’s disease (HD). A growing body of evidence has indicated that motoric and cognitive recovery can be achieved following transplantation of functional striatal precursor cells into the damaged animal and human striatum. However, currently no one cell source is suitable for widespread and long term clinical use. Human induced pluripotent stem cells (iPSC) can theoretically be generated from any viable human cells and are logistically suitable for CRT. Additionally, previous work has demonstrated iPSCs may retain former epigenetic mechanisms that control gene expression and promote a return to previously held cell fate. If generated from suitable tissue sources, this ‘epigenetic memory’ may facilitate improved generation of functional striatal precursor cells compared to other logistically suitable cell sources. Aim This work aims to identify the potential of fetal striatal-derived iPSC epigenetic memory to determine functional MSN differentiation, and to explore the potential of this cell source to induce recovery in an animal model of HD. Experimental plans To achieve these aims we have generated iPSC lines from fetal striatal, cortical and fibroblast tissues. These lines are currently being characterised in vitro for pluripotency, and MSN generation potential, directly comparing the different tissue derived iPSC lines with embryonic stem cell (ESC) controls. We intend to characterise DNA methylation profiles of each iPSC line across the induction and differentiation process, to gain insight into the epigenetic profiles at key stages. Lastly, in vivo graft characterisation and behavioural assessments will determine if the presence of epigenetic memory in fetal striatal-derived iPSCs directed towards a neural precursor fate can be used to generate better functioning grafts than those derived from ESC and primary fetal cells.

Background Although Huntington’s disease (HD) affects multiple brain areas it has a disproportionate and early effect on the striatal medium spiny neurons (MSNs). Transplantation of developing rat MSNs, derived from embryonic day 14 (E14) whole ganglionic eminence (WGE), into a rodent lesion models of HD results in grafts that integrate into the host striatum, alleviating both motor and cognitive symptoms. The equivalent gestational period for human fetal WGE is thought to be 8–10 weeks post conception (pc), but transplants of tissue in this range are more morphologically and functionally variable. One potential explanation is that the generally accepted age range for human cells is not in fact optimal. Aims To estimate the optimal age of donor tissue for transplantation in HD, based on molecular, histological and electrophysiological comparisons of human and mouse WGE development across the period of peak MSN development. Methods We compared WGE from mouse (E12 to E16) and human (6–12 weeks pc) using QPCR, in situ hybridisation of tissue sections and in vitro immunocytochemistry for a range of known and potential striatal markers, and also plated cells for electrophysiology. Results Developmentally regulated striatal markers, such as GSX2, CTIP and FOXP1, show delayed expression in the human compared to rodent WGE, suggesting that human WGE within this age range is less developmentally mature than previously appreciated. This is supported by the findings that human WGE cells are still proliferative at 11 weeks pc and appear electrophysiologically less mature compared to the mouse cells. Conclusion These results suggest that the gestational range used to date for human WGE may be insufficiently mature for grafts to develop optimally. This information could help to identify optimal age of fetal donor tissue and act as a reference for MSN differentiation from pluripotent stem cells, but will need verification following transplantation.