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Huntington's disease (HD) is a debilitating, genetically inherited neurodegenerative disorder that results in early loss of medium spiny neurons from the striatum and subsequent degeneration of cortical and other subcortical brain regions. Behavioral changes manifest as a range of motor, cognitive, and neuropsychiatric impairments. It has been established that replacement of the degenerated medium spiny neurons with rat-derived fetal whole ganglionic eminence (rWGE) tissue can alleviate motor and cognitive deficits in preclinical rodent models of HD. However, clinical application of this cell replacement therapy requires the use of human-derived (hWGE), not rWGE, tissue. Despite this, little is currently known about the functional efficacy of hWGE. The aim of this study was to directly compare the ability of the gold standard rWGE grafts, against the clinically relevant hWGE grafts, on a range of behavioral tests of motor function. Lister hooded rats either remained as unoperated controls or received unilateral excitotoxic lesions of the lateral neostriatum. Subsets of lesioned rats then received transplants of either rWGE or hWGE primary fetal tissue into the lateral striatum. All rats were tested postlesion and postgraft on the following tests of motor function: staircase test, apomorphine-induced rotation, cylinder test, adjusting steps test, and vibrissae-evoked touch test. At 21 weeks postgraft, brain tissue was taken for histological analysis. The results revealed comparable improvements in apomorphine-induced rotational bias and the vibrissae test, despite larger graft volumes in the hWGE cohort. hWGE grafts, but not rWGE grafts, stabilized behavioral performance on the adjusting steps test. These results have implications for clinical application of cell replacement therapies, as well as providing a foundation for the development of stem cell-derived cell therapy products.

With the use of cell replacement therapies as a realistic prospect for conditions such as Parkinson's and Huntington's diseases, the logistics of the delivery of cell suspensions to deep brain targets is a topic for consideration. Because of the large cannulae required for such procedures, we need to consider the behavior of cell suspensions within the cannulae if we are to ensure that the injected cells are distributed as intended within the target tissue. We have investigated the behavior of primary embryonic cell suspensions of neural tissue, in cannulae of different diameters, using a protocol designed to mimic the handling and injection of cells during clinical application. Internal cannula diameter had a large effect on the distribution of cells during their dispensation from the syringe. In vertical or near vertical cannulae, cells settled toward the tip of the needle, and were dispensed unevenly, with the majority of cells emerging in the first 10-20% of the injectate. In horizontal or near-horizontal cannulae, we observed the opposite effect, such that few cells were dispensed in the first 80% of the injectate, and the majority emerged in the final 10-20%. Use of a glass cannula showed that the results obtained using the horizontal cannula were caused by settling and adherence of the cells on the side of the cannulae, such that during dispensation, the overlying, cell-free solution was dispensed first, prior to the emergence of the cells. We show that the behavior of cells in such cannulae is affected by the cannula diameter, and by the material of the cannula itself. In horizontal cannulae, uneven expulsion of cells from the needle can be ameliorated by regular rotation of the cannula during the procedure. We discuss the potential impact of these observations on the translation of cell therapies to the clinic.

"Proof-of-principle" that cell replacement therapy works for neurodegeneration has been reported, but only using donor cells collected from fetal brain tissue obtained from surgical terminations of pregnancy. Surgical terminations of pregnancy represent an increasingly limited supply of donor cells due to the tendency towards performing medical termination in much of Europe. This imposes a severe constraint on further experimental and clinical cell transplantation research. Therefore, we explore here the feasibility of using medical termination tissue as a donor source. Products of conception were retrieved from surgical terminations over the last 7 years and from medical terminations over the last 2.5 years. The number of collections that yielded fetal tissue, viable brain tissue, and identifiable brain regions (ganglionic eminence, ventral mesencephalon, and neocortex) were recorded. We studied cell viability, cell physiological properties, and differentiation potential both in vitro and following transplantation into the central nervous system of rodent models of neurodegenerative disease. Within equivalent periods, we were able to collect substantially greater numbers of fetal remains from medical than from surgical terminations of pregnancy, and the medical terminations yielded a much higher proportion of identifiable and dissectible brain tissue. Furthermore, we demonstrate that harvested cells retain the capacity to differentiate into neurons with characteristics appropriate to the region from which they are dissected. We show that, contrary to widespread assumption, medical termination of pregnancy-derived fetal brain cells represent a feasible and more readily available source of human fetal tissue for experimental cell transplantation with the potential for use in future clinical trials in human neurodegenerative disease.

Obtaining accurately staged rat embryos can be difficult because of the variety of breeding protocols employed and because precise staging cannot be confirmed until excision of the embryos from the dam. The detection of estrus, pairing of animals, and confirmation of pregnancies is generally left to commercial suppliers, as in-house breeding can be laborious and unpredictable. Here we describe a simple, reliable in-house breeding protocol for the generation of accurately staged embryos as assessed by measurements of average crown to rump length (CRL).

In rat models of Parkinson's and Huntington's diseases, embryonic neural cells obtained from embryos of specified ages can be implanted into the brain to partially restore both physiology and function. However, in litters produced using overnight mating protocols (often from commercial suppliers), the embryonic age can be difficult to determine precisely. As a result, embryonic size based on crown to rump length (CRL) is usually a more reliable method of embryo staging than the day of mating. This approach is not without difficulty. There are a number of rat staging scales in the literature, none of which deal with donor ages younger than E13, and there are discrepancies between scales at some donor ages. In the present article, we have devised a short mating-period protocol to produce precisely aged embryos. We show that CRL is a highly accurate, reproducible index of donor age and we present an updated embryonic staging scale for Sprague-Dawley (CD) rats that includes donor ages younger than those previously reported.

Only a small proportion of dopamine neurons in nigral grafts typically survive transplantation into the adult striatum. Since many anaesthetics reduce blood flow and disturb a variety of brain metabolites, surgical anaesthesia may be one of the factors that compromise graft survival. Conversely, the lowered core body temperature induced by some anaesthetics might promote the survival of grafted cells by slowing their metabolism. In an initial screen, the widely-used surgical anaesthetic, equithesin, was found to reduce core temperature, mean arterial blood pressure, and to increase the partial pressure of oxygen in arterial blood without producing any significant alteration in arterial pH or the partial pressure of carbon dioxide. In the main experiment, rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal bundle received dopamine-rich embryonic nigral grafts injected into the deafferented neostriatum via previously implanted guide cannulae, which allowed comparison to be made of graft survival after transplantation into awake and in re-anaesthetised animals. There were no significant differences between groups in either the functional effects of the grafts to compensate amphetamine-induced rotation, or in the survival and growth of the grafts as measured in post mortem histology. We therefore conclude that anaesthesia per se is not a major contributory factor in the relatively poor survival of dopamine neurons following transplantation into the rat striatum.

The study examined the importance of embryonic donor age for the survival of nigral grafts in 6-OHDA-lesioned marmosets. The issue as to whether donor age is critical for the survival of nigral grafts in primates is controversial, because several early reports suggested that relatively old tissue could survive transplantation and produce functional benefits in monkeys, in contrast to the restrictive time dependence observed in rodents. Embryonic marmoset donors embryos of three different ages were employed: 1) E74 (Carnegie stage 18-19); 2) E83-84 (Carnegie stage 23+); 3) E92-93 (foetal period). The nigral neurons derived from the ventral mesencephalon in the two older donor age groups did not survive well when grafted to the striatum of adult marmosets with unilateral 6-OHDA lesions. Although a few tyrosine hydroxylase (TH+) neurons could be identified by immunohistochemistry at graft sites in all recipients in older donor age groups, the numbers of surviving neurons in these were small, on average typically less than 100 TH+ cells. These small grafts were not sufficient to affect amphetamine-induced rotation. In contrast, many more TH+ cells typically survived transplantation in the recipients of graft tissue derived from the youngest donors and amphetamine-induced rotation was significantly reduced in this group alone. The time course and extent of the reduction in rotation was remarkably similar to that observed in previous marmoset nigral graft studies, confirming the utility of amphetamine-induced rotation as a sensitive and reliable indicator of nigral graft function in this species. Considering these results and other recent evidence from monkey to monkey, human to rat, and human to human graft studies, the survival of embryonic nigral tissues derived from primate donors transplanted into the striatum does appear to be critically dependent on the age of the donor tissue.