Single-cell RNAseq identifies clonally expanded antigen-specific T-cells following intradermal injection of gold nanoparticles loaded with diabetes autoantigen in humans.

Gold nanoparticles (GNPs) have been used in the development of novel therapies as a way of delivery of both stimulatory and tolerogenic peptide cargoes. Here we report that intradermal injection of GNPs loaded with the proinsulin peptide C19-A3, in patients with type 1 diabetes, results in recruitment and retention of immune cells in the skin. These include large numbers of clonally expanded T-cells sharing the same paired T-cell receptors (TCRs) with activated phenotypes, half of which, when the TCRs were re-expressed in a cell-based system, were confirmed to be specific for either GNP or proinsulin. All the identified gold-specific clones were CD8+, whilst proinsulin-specific clones were both CD8+ and CD4+. Proinsulin-specific CD8+ clones had a distinctive cytotoxic phenotype with overexpression of granulysin (GNLY) and KIR receptors. Clonally expanded antigen-specific T cells remained in situ for months to years, with a spectrum of tissue resident memory and effector memory phenotypes. As the T-cell response is divided between targeting the gold core and the antigenic cargo, this offers a route to improving resident memory T-cells formation in response to vaccines. In addition, our scRNAseq data indicate that focusing on clonally expanded skin infiltrating T-cells recruited to intradermally injected antigen is a highly efficient method to enrich and identify antigen-specific cells. This approach has the potential to be used to monitor the intradermal delivery of antigens and nanoparticles for immune modulation in humans.

Induced pluripotent stem cells derived from the developing striatum as a potential donor source for cell replacement therapy for Huntington disease.

BACKGROUND: 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. RESULTS: 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.

Direct Comparison of Rat- and Human-Derived Ganglionic Eminence Tissue Grafts on Motor Function.

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.

Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell-derived medium spiny neurons.

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.

Behavioural recovery on simple and complex tasks by means of cell replacement therapy in unilateral 6-hydroxydopamine-lesioned mice.

Before cell replacement therapies can enter the clinic, it is imperative to test the therapeutic benefits in well-described animal models. In the present study, we aimed to investigate the effects of 6-hydroxydopamine lesions to the medial forebrain bundle and subsequent grafting of embryonic day (E)12.5 ventral mesencephalon into the denervated striatum in C57/Bl6 mice on a battery of simple motor tests (drug-induced rotation, rotarod, and corridor) and the lateralised choice reaction time task conducted in the mouse nine-hole box. Histological analysis confirmed effective lesions and good graft survival. The lesion induced marked deficits in the choice reaction time task, the rotarod test, and corridor test, and these deficits were partially but significantly alleviated in the grafted mice. Although the lesions induced significant rotation following injections of amphetamine and apomorphine, respectively, the grafts did not, suprisingly, alleviate the rotation deficit. This study shows the ability of ventral mesencephalic tissue to ameliorate some of the lesion-induced deficits, and the power of operant testing in detecting small but significant improvements. The behavioural tests presented are useful drug-free approaches for evaluating cell-based therapies.

Amphetamine-induced dyskinesia in the transplanted hemi-Parkinsonian mouse.

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.