Directed differentiation of mouse embryonic stem cells for transplantation in Huntington's disease

The research presented in this thesis focused on neural precursors derived from mouse ES cells (also called neural embryoid bodies, NEBs) in particular attempting to direct their differentiation towards specific telencephalic precursors, with a view to providing an alternative donor cell source to primary foetal tissue for the generation of DARPP-32 positive medium spiny neurons (MSNs) for neural transplantation in Huntington's disease. Neural induction of mouse ES cells was addressed, with further directed differentiation by addition of factors known to be important in the neural tube for development of specific telencephalic precursors. Subsequent neuronal differentiation of precursors was assessed both in vitro and in vivo. Chapter 3 characterised the neural and forebrain features of NEBs up to day 8 in neural induction medium, using a reporter cell line, in which LacZ was tagged to the early forebrain marker, Foxgl. Addition of FGF2 but not the Wnt inhibitor DKK-1, increased the forebrain population within the overall cell population. Day 8 NEBs, subjected to neuronal differentiation conditions, yielded p-III-tubulin positive neurons but no DARPP-32 expression. In Chapter 4, NEBs cultured for either 8 or 16 days were compared for expression of neural markers and then transplanted into the QA lesioned rat striatum to assess their potential to generate mature phenotypes in vivo. Day 8 NEBs generated teratomas by 2 weeks post transplantation. At 6 weeks post-transplantation, day 16 NEBs generated heterogeneous grafts with graft-derived neurons and glia, but no graft-derived DARPP-32 expression. Expression of dorso-ventral telencephalon markers in NEBs was investigated in Chapter 5, drawing comparisons to expression in the mouse (El4) developing telencephalon. The effects of the SHH agonist, purmorphamine, and the SHH antagonist, cyclopamine, were used in an attempt to enrich the forebrain precursors generated in Chapter 3 for ventral telencephalic-like precursors. Although gene expression changes were demonstrated, no distinct dorso-ventral patterns were shown. In vitro neuronal differentiation of day 16 NEBs was analysed in Chapter 6, in particular looking for expression of the MSN marker DARPP-32. Cultures of cells from the lateral ganglionic eminence (LGE) (the origin of MSNs) were used to determine if certain factors could increase the yield of DARPP-32 positive neurons. Addition of BDNF yielded the highest proportion of DARPP-32 from LGE. However, when this condition was applied to neuronal differentiation cultures of day 16 NEBs there was no effect on the expression of GAB A, DARPP-32 or FoxPl, all markers of MSNs. In Chapter 7, day 16 NEBs, with and without addition of purmorphamine, were transplanted into the QA lesioned mouse striatum. All resulting grafts were small, but analysis revealed densities of neuronal and striatal markers in day 16 NEB-derived grafts to be similar to E14-derived grafts

Molecular regulation of striatal development: a review

The central nervous system is composed of the brain and the spinal cord. The brain is a complex organ that processes and coordinates activities of the body in bilaterian, higher-order animals. The development of the brain mirrors its complex function as it requires intricate genetic signalling at specific times, and deviations from this can lead to brain malformations such as anencephaly. Research into how the CNS is specified and patterned has been studied extensively in chick, fish, frog, and mice, but findings from the latter will be emphasised here as higher-order mammals show most similarity to the human brain. Specifically, we will focus on the embryonic development of an important forebrain structure, the striatum (also known as the dorsal striatum or neostriatum). Over the past decade, research on striatal development in mice has led to an influx of new information about the genes involved, but the precise orchestration between the genes, signalling molecules, and transcription factors remains unanswered. We aim to summarise what is known to date about the tightly controlled network of interacting genes that control striatal development. This paper will discuss early telencephalon patterning and dorsal ventral patterning with specific reference to the genes involved in striatal development

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

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

Medical terminations of pregnancy: A viable source of tissue for cell replacement therapy for neurodegenerative disorders

“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

Influenza and associated co-infections in critically ill immunosuppressed patients

Abstract Background It is unclear whether influenza infection and associated co-infection are associated with patient-important outcomes in critically ill immunocompromised patients with acute respiratory failure. Methods Preplanned secondary analysis of EFRAIM, a prospective cohort study of 68 hospitals in 16 countries. We included 1611 patients aged 18 years or older with non-AIDS-related immunocompromise, who were admitted to the ICU with acute hypoxemic respiratory failure. The main exposure of interest was influenza infection status. The primary outcome of interest was all-cause hospital mortality, and secondary outcomes ICU length of stay (LOS) and 90-day mortality. Results Influenza infection status was categorized into four groups: patients with influenza alone (n = 95, 5.8%), patients with influenza plus pulmonary co-infection (n = 58, 3.6%), patients with non-influenza pulmonary infection (n = 820, 50.9%), and patients without pulmonary infection (n = 638, 39.6%). Influenza infection status was associated with a requirement for intubation and with LOS in ICU (P < 0.001). Patients with influenza plus co-infection had the highest rates of intubation and longest ICU LOS. On crude analysis, influenza infection status was associated with ICU mortality (P < 0.001) but not hospital mortality (P = 0.09). Patients with influenza plus co-infection and patients with non-influenza infection alone had similar ICU mortality (41% and 37% respectively) that was higher than patients with influenza alone or those without infection (33% and 26% respectively). A propensity score-matched analysis did not show a difference in hospital mortality attributable to influenza infection (OR = 1.01, 95%CI 0.90–1.13, P = 0.85). Age, severity scores, ARDS, and performance status were all associated with ICU, hospital, and 90-day mortality. Conclusions Category of infectious etiology of respiratory failure (influenza, non-influenza, influenza plus co-infection, and non-infectious) was associated with ICU but not hospital mortality. In a propensity score-matched analysis, influenza infection was not associated with the primary outcome of hospital mortality. Overall, influenza infection alone may not be an independent risk factor for hospital mortality in immunosuppressed patients

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

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