Challenges in progressing cell therapies to the clinic for Huntington's disease: A review of the progress made with pluripotent stem cell derived medium spiny neurons.

Huntington's disease (HD) is a hereditary, neurodegenerative disorder characterized by a triad of symptoms: motor, cognitive and psychiatric. HD is caused by a genetic mutation, expansion of the CAG repeat in the huntingtin gene, which results in loss of medium spiny neurons (MSNs) of the striatum. Cell replacement therapy (CRT) has emerged as a possible therapy for HD, aiming to replace those cells lost to the disease process and alleviate its symptoms. Initial pre-clinical studies used primary fetal striatal cells to provide proof-of-principal that CRT can bring about functional recovery on some behavioral tasks following transplantation into HD models. Alternative donor cell sources are required if CRT is to become a viable therapeutic option and human pluripotent stem cell (hPSC) sources, which have undergone differentiation toward the MSNs lost to the disease process, have proved to be strong candidates. The focus of this chapter is to review work conducted on the functional assessment of animals following transplantation of hPSC-derived MSNs. We discuss different ways that graft function has been assessed, and the results that have been achieved to date. In addition, this chapter presents and discusses challenges that remain in this field.

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.

Dopaminergic Progenitors Derived From Epiblast Stem Cells Function Similarly to Primary VM-Derived Progenitors When Transplanted Into a Parkinson's Disease Model.

Neural transplantation in neurodegenerative diseases such as Parkinson's disease (PD) offers to replace cells lost during the progression of the disease process. Primary fetal ventral mesencephalon (VM), the origin of bona fide midbrain dopaminergic (DAergic) precursors, is currently the gold standard source of cells for transplantation in PD. However, the use of tissue from this source raises ethical and logistical constraints necessitating the need for alternative supplies of donor cells. The requirement of any alternative donor cell source is to have the capability to generate authentic mature DAergic neurons, which could be utilized in cell-replacement strategies. Mouse pluripotent stem cells can efficiently generate electrochemically mature midbrain DAergic precursors in vitro using a stepwise control of FGF signaling. Here, we have compared DAergic transplants derived from two progenitor cell sources in an allograft system: mouse epiblast stem cells (EpiSC) and primary fetal mouse VM tissue. Cells were transplanted into the striatum of 6-OHDA lesioned mice pre-treated with L-DOPA. Drug-induced rotations, a number of motor tests and drug-induced abnormal involuntary movements (AIMs) were assessed. Functional improvements were demonstrated post-transplantation in some behavioral tests, with no difference in graft volume or the number of TH immuno-positive cells in the grafts of the two transplant groups. L-DOPA-induced AIMs and amphetamine-induced AIMs were observed in both transplant groups, with no differences in rate or severity between the two groups. Collectively, in this mouse-to-mouse allograft system, we report no significant differences in the functional ability between the gold standard primary VM derived and pluripotent stem cell-derived DAergic transplants.

Dissection and Preparation of Human Primary Fetal Ganglionic Eminence Tissue for Research and Clinical Applications.

Here, we describe detailed dissection and enzymatic dissociation protocols for the ganglionic eminences from the developing human brain to generate viable quasi-single cell suspensions for subsequent use in transplantation or cell culture. These reliable and reproducible protocols can provide tissue for use in the study of the developing human brain, as well as for the preparation of donor cells for transplantation in Huntington's disease (HD). For use in the clinic as a therapy for HD, the translation of these protocols from the research laboratory to the GMP suite is described, including modification to reagents used and appropriate monitoring and tissue release criteria.

Is there a place for human fetal-derived stem cells for cell replacement therapy in Huntington's disease?

Huntington's disease (HD) is a neurodegenerative disease that offers an excellent paradigm for cell replacement therapy because of the associated relatively focal cell loss in the striatum. The predominant cells lost in this condition are striatal medium spiny neurons (MSNs). Transplantation of developing MSNs taken from the fetal brain has provided proof of concept that donor MSNs can survive, integrate and bring about a degree of functional recovery in both pre-clinical studies and in a limited number of clinical trials. The scarcity of human fetal tissue, and the logistics of coordinating collection and dissection of tissue with neurosurgical procedures makes the use of fetal tissue for this purpose both complex and limiting. Alternative donor cell sources which are expandable in culture prior to transplantation are currently being sought. Two potential donor cell sources which have received most attention recently are embryonic stem (ES) cells and adult induced pluripotent stem (iPS) cells, both of which can be directed to MSN-like fates, although achieving a genuine MSN fate has proven to be difficult. All potential donor sources have challenges in terms of their clinical application for regenerative medicine, and thus it is important to continue exploring a wide variety of expandable cells. In this review we discuss two less well-reported potential donor cell sources; embryonic germ (EG) cells and fetal neural precursors (FNPs), both are which are fetal-derived and have some properties that could make them useful for regenerative medicine applications.

Can manipulation of differentiation conditions eliminate proliferative cells from a population of ES cell-derived forebrain cells?

There is preliminary evidence that implantation of primary fetal striatal cells provides functional benefit in patients with Huntington's disease, a neurodegenerative condition resulting in loss of medium-sized spiny neurons (MSN) of the striatum. Scarcity of primary fetal tissue means it is important to identify a renewable source of cells from which to derive donor MSNs. Embryonic stem (ES) cells, which predominantly default to telencephalic-like precursors in chemically defined medium (CDM), offer a potentially inexhaustible supply of cells capable of generating the desired neurons. Using an ES cell line, with the forebrain marker FoxG1 tagged to the LacZ reporter, we assessed effects of known developmental factors on the yield of forebrain-like precursor cells in CDM suspension culture. Addition of FGF2, but not DKK1, increased the proportion of FoxG1-expressing cells at day 8 of neural induction. Oct4 was expressed at day 8, but was undetectable by day 16. Differentiation of day 16 precursors generated GABA-expressing neurons, with few DARPP32 positive MSNs. Transplantation of day 8 precursor cells into quinolinic acid-lesioned striata resulted in generation of teratomas. However, transplantation of day 16 precursors yielded grafts expressing neuronal markers including NeuN, calbindin and parvalbumin, but no DARPP32 6 weeks post-transplantation. Manipulation of fate of ES cells requires optimization of both concentration and timing of addition of factors to culture systems to generate the desired phenotypes. Furthermore, we highlight the value of increasing the precursor phase of ES cell suspension culture when directing differentiation toward forebrain fate, so as to dramatically reduce the risk of teratoma formation.

Activin A directs striatal projection neuron differentiation of human pluripotent stem cells.

The efficient generation of striatal neurons from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) is fundamental for realising their promise in disease modelling, pharmaceutical drug screening and cell therapy for Huntington's disease. GABAergic medium-sized spiny neurons (MSNs) are the principal projection neurons of the striatum and specifically degenerate in the early phase of Huntington's disease. Here we report that activin A induces lateral ganglionic eminence (LGE) characteristics in nascent neural progenitors derived from hESCs and hiPSCs in a sonic hedgehog-independent manner. Correct specification of striatal phenotype was further demonstrated by the induction of the striatal transcription factors CTIP2, GSX2 and FOXP2. Crucially, these human LGE progenitors readily differentiate into postmitotic neurons expressing the striatal projection neuron signature marker DARPP32, both in culture and following transplantation in the adult striatum in a rat model of Huntington's disease. Activin-induced neurons also exhibit appropriate striatal-like electrophysiology in vitro. Together, our findings demonstrate a novel route for efficient differentiation of GABAergic striatal MSNs from human pluripotent stem cells.

Components of the endocannabinoid and dopamine systems are dysregulated in Huntington's disease: analysis of publicly available microarray datasets.

The endocannabinoid system (ECS) and the dopaminergic system (DAS) are two major regulators of basal ganglia function. During Huntington's disease (HD) pathogenesis, the expression of genes in both the ECS and DAS is dysregulated. The purpose of this study was to determine the changes that were consistently observed in the ECS and DAS during HD progression in the central nervous system (CNS) and in the periphery in different models of HD and human HD tissue. To do this, we conducted a meta-analysis of differential gene expression in the ECS and DAS using publicly available microarray data. The consolidated data were summarized as observed changes in gene expression (OCGE) using a weighted sum for each gene. In addition, consolidated data were compared to previously published studies that were not available in the gene expression omnibus (GEO) database. The resulting data confirm gene expression changes observed using different approaches and provide novel insights into the consistency between changes observed in human tissue and various models, as well as disease stage- and tissue-specific transcriptional dysregulation in HD. The major implication of the systems-wide data presented here is that therapeutic strategies targeting the ECS or DAS must consider the dynamic changes in gene expression over time and in different body areas, which occur during HD progression and the interconnectedness of the two systems.

Is the adult mouse striatum a hostile host for neural transplant survival?

Human donor cells, including neurally directed embryonic stem cells and induced pluripotent stem cells with the potential to be used for neural transplantation in a range of neurodegenerative disorders, must first be tested preclinically in rodent models of disease to demonstrate safety and efficacy. One strategy for circumventing the rejection of xenotransplanted human cells is to desensitize the host animal to human cells in the early neonatal period so that a subsequent transplant in adulthood is not immunorejected. This method has been robustly validated in the rat, but currently not in the mouse in which most transgenic models of neurodegeneration have been generated. Thus, we set out to determine whether this could be achieved through modification of the existing rat protocol. Mice were inoculated in the neonatal period with a suspension of human embryonic cortical tissue of varying cell numbers, and received a subsequent human embryonic cortical tissue cell transplant in adulthood. Graft survival was compared with those in mice immunosuppressed with cyclosporine A and those receiving allografts of mouse whole ganglionic eminence tissue. Poor survival was found across all groups, suggesting a general problem with the use of mouse hosts for testing human donor cells.

Producing striatal phenotypes for transplantation in Huntington's disease.

Neural transplantation as a therapeutic strategy in neurodegenerative disorders offers to replace cells lost during the disease process, with the potential to reconstruct dysfunctional circuitry, thus alleviating associated disease symptoms. The focal loss of striatal cells, specifically medium-sized spiny neurons (MSN) in Huntington's disease (HD), makes transplantation a therapeutic option. Here, we review the progress made in generating striatal MSN phenotypes for transplantation in HD. We discuss the use of primary fetal tissue as a donor source in both preclinical and clinical studies and assess the options for renewable cell sources. We evaluate progress in directing the differentiation of renewable cells towards a striatal MSN phenotype for HD.

Long-term expansion of human foetal neural progenitors leads to reduced graft viability in the neonatal rat brain.

We previously reported that early passage human foetal neural progenitors (hFNPs) survive long-term in the rodent host brain whereas late passage cells disappear at later post-graft survival times. The extent to which this finding is related to changes in the expanded FNPs or in the adult host brain environment was not determined. Here we report the effect of expanding hFNPs for different periods of time in vitro on their ability to survive transplantation into the neonatal rat hippocampus, a generally more permissive environment than the adult rat brain. After 2 and 8 weeks in vitro, transplanted hFNPs formed large grafts, most of which survived well until at least 12 weeks. However, following continued expansion, hFNPs formed smaller grafts, and cells transplanted after 20 weeks expansion produced no surviving grafts, even at early survival times. To determine whether this could be due to a dilution of "true" neural stem cells through more differentiated progeny over time in culture, we derived homogeneous neural stem (NS) cells grown as a monolayer from the 8 week expanded hFNPs. These cells homogeneously expressed the neural stem cell markers sox-2, 3CB2 and nestin and were expanded for 5 months before transplantation into the neonatal rat brain. However, these cells exhibited a similar survival profile to the long-term expanded FNPs. These results indicate that, while the cellular phenotype of neural stem cells may appear to be stable in vitro using standard markers, expansion profoundly influences the ability of such cells to form viable grafts.

Neonatal desensitization allows long-term survival of neural xenotransplants without immunosuppression.

Preclinical development of human cells for potential therapeutic application in neurodegenerative diseases requires that their long-term survival, stability and functional efficacy be studied in animal models of human disease. Here we describe a strategy for long-term immune protection of human fetal and stem cell-derived neural cells transplanted into the adult rat brain, by desensitizing the host rat to similar cells in the neonatal period, without the need for additional immunosuppression.

Striatal graft projections are influenced by donor cell type and not the immunogenic background.

Reconstruction of CNS circuitry is a major aim of neural transplantation, and is currently being assessed clinically using foetal striatal tissue in Huntington's disease. Recent work suggests that neuronal precursors derived from foetal striatum may have a greater capacity than primary foetal striatum to project to the usual striatal target areas such as the globus pallidus and substantia nigra, raising the possibility that they have a greater potential for circuit reconstruction. However, comparing the reconstructive capacity of the two donor cells types is confounded by the fact that many precursor experiments have been carried out in a xenogeneic background in order to utilize species-specific markers for tracking the donor cells, whereas most primary foetal transplant studies have utilized an allograft paradigm. Thus, differences in immunogenic background could influence the findings; for example, xenogeneic grafts may not recognize host inhibitory signals, thereby encouraging more profuse and extensive projections. We have addressed this issue directly by comparing foetal neural precursor and primary foetal grafts in both allo- and xenograft environments using several labelling techniques, including GFP-transgenic mice and LacZ-labelled cells as donor tissue and iontophoretic injection of the anterograde tracers BDA, neurobiotin and PHA-L in the host. We present clear evidence that foetal neural precursors produce grafts with richer axonal outgrowth than primary foetal grafts, and that this is independent of the immunogenic background. Furthermore, both neural precursor and primary grafts derived from human foetal tissue produced a significantly richer outgrowth than do grafts of mouse donor tissue, which may relate to their large final graft volume and the greater intrinsic potential of human CNS neurons for greater axon elongation.