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Background\ud Cell replacement therapy (CRT) for Huntington disease (HD) requires a source of striatal (STR) progenitors capable of restoring the function lost due to STR degeneration. Authentic STR progenitors can be collected from the fetal putative striatum, or whole ganglionic eminence (WGE), but these tissues remain impractical for widespread clinical application, and alternative donor sources are required. Here we begin exploring the possibility that induced pluripotent stem cells (iPSC) derived from WGE may retain an epigenetic memory of their tissue of origin, which could enhance their ability to differentiate into STR cells.\ud Results\ud We generate four iPSC lines from human WGE (hWGE) and establish that they have a capacity similar to human embryonic stem cells with regard to their ability to differentiate toward an STR phenotype, as measured by expression and demethylation of key STR genes, while maintaining an overall different methylome. Finally, we demonstrate that these STR-differentiated hWGE iPSCs share characteristics with hWGE (i.e., authentic STR tissues) both in vitro and following transplantation into an HD model. Overall, iPSCs derived from human WGE show promise as a donor source for CRT for HD.

Kv7 channels determine the resting membrane potential of neurons and regulate their excitability. Even though dysfunction of Kv7 channels has been linked to several debilitating childhood neuronal disorders, the ontogeny of the constituent genes, which encode Kv7 channels (KNCQ), and expression of their subunits have been largely unexplored. Here, we show that developmentally regulated expression of specific KCNQ mRNA and Kv7 channel subunits in mouse and human striatum is crucial to the functional maturation of mouse striatal neurons and human-induced pluripotent stem cell-derived neurons. This demonstrates their pivotal role in normal development and maturation, the knowledge of which can now be harnessed to synchronise and accelerate neuronal differentiation of stem cell-derived neurons, enhancing their utility for disease modelling and drug discovery.

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.

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.

Neural cells isolated from the brain have a number of research and clinical applications, including transplantation to patients with neurodegenerative conditions. Tissue supply is one of the major limiting factors to clinical transplantation. Cryopreservation of primary neural cells would improve supply, aid in organisation of transplantation surgery and facilitate research. To date, cryopreservation using standard methods has resulted in reduced yield and/or viability of primary neural tissue. In order to optimise freezing protocols specifically for such cells, the non-osmotic volume (V(b)), water permeability (L(p)) and permeability to cryoprotectant (P(cpa)) were determined. Murine foetal brain tissue from the ganglionic eminence (GE), ventral mesencephalon (VM), or neocortical mantle (Ctx) was trypsinised to a single cell suspension. To determine V(b,) cell volume was measured after exposure to anisotonic solutions of sucrose (150-1500 mOsmol/kg). L(p) (mum/min.atm) and P(cpa) (mum/s) were determined for GE cells by measuring cell volume during exposure to 1.5 mol/l cryoprotectant. Cell volume was determined using an electronic particle counting method. V(b) was 27% for Ctx and GE, and 30% for VM. The osmotic response of GE cells was similar in the presence of propane-1,2-diol and dimethyl sulphoxide. In the presence of ethylene glycol, cell volume decrease was greater on initial exposure to cryoprotectant and recovery slower. Differences in L(p,) but not P(cpa), were found between cryoprotectants. The present results provide key parameters for optimisation of freezing protocols for cryopreservation of primary foetal brain tissues for application in neural cell transplantation.

Synaptic transmission depends on the regulated surface expression of neurotransmitter receptors, but many of the cellular processes required to achieve this remain poorly understood. To better define specific mechanisms for the GABA(B) receptor (GABA(B)R) trafficking, we screened for proteins that bind to the carboxy-terminus of the GABA(B1) subunit. We report the identification and characterization of a novel 130-kDa protein, GPCR interacting scaffolding protein (GISP), that interacts directly with the GABA(B1) subunit via a coiled-coil domain. GISP co-fractionates with GABA(B)R and with the postsynaptic density and co-immunoprecipitates with GABA(B1) and GABA(B2) from rat brain. In cultured hippocampal neurons, GISP displays a punctate dendritic distribution and has an overlapping localization with GABA(B)Rs. When co-expressed with GABA(B)Rs in human embryonic kidney cells, GISP promotes GABA(B)R surface expression and enhances both baclofen-evoked extracellular signal-regulated kinase (ERK) phosphorylation and G-protein inwardly rectifying potassium channel (GIRK) currents. These results suggest that GISP is involved in the forward trafficking and stabilization of functional GABA(B)Rs.

A systematic characterization of the spatio-temporal gene expression during human neurodevelopment is essential to understand brain function in both physiological and pathological conditions. In recent years, stem cell technology has provided an in vitro tool to recapitulate human development, permitting also the generation of human models for many diseases. The correct differentiation of human pluripotent stem cell (hPSC) into specific cell types should be evaluated by comparison with specific cells/tissue profiles from the equivalent adult in vivo organ. Here, we define by a quantitative high-throughput gene expression analysis the subset of specific genes of the whole ganglionic eminence (WGE) and adult human striatum. Our results demonstrate that not only the number of specific genes is crucial but also their relative expression levels between brain areas. We next used these gene profiles to characterize the differentiation of hPSCs. Our findings demonstrate a temporal progression of gene expression during striatal differentiation of hPSCs from a WGE toward an adult striatum identity. Present results establish a gene expression profile to qualitatively and quantitatively evaluate the telencephalic hPSC-derived progenitors eventually used for transplantation and mature striatal neurons for disease modeling and drug-screening.