Amanda-Jayne Carr | University College London (original) (raw)

Papers by Amanda-Jayne Carr

Retinal degeneration represents a huge burden of blinding disease, and currently there are no eff... more Retinal degeneration represents a huge burden of blinding disease, and currently there are no effective treatments that reverse the most common causes of neural retinal degeneration. Stem cell biology has the potential to significantly ease this burden, not only through the development of disease models of retinal degeneration but also in the manufacture of a replacement for the neural retinal tissue. This review summarizes the major advancements in the last decade in the field of neural retinal regeneration with an emphasis on the differentiation of embryonic and induced pluripotent stem cells into cells with retinal and specifically photoreceptor characteristics.

Stem cell therapy for retinal disease is under way, and several clinical trials are currently rec... more Stem cell therapy for retinal disease is under way, and several clinical trials are currently recruiting. These trials use human embryonic, foetal and umbilical cord tissue-derived stem cells and bone marrow-derived stem cells to treat visual disorders such as age-related macular degeneration, Stargardt's disease and retinitis pigmentosa. Over a decade of analysing the developmental cues involved in retinal generation and stem cell biology, coupled with extensive surgical research, have yielded differing cellular approaches to tackle these retinopathies. Here, we review these various stem cell-based approaches for treating retinal diseases and discuss future directions and challenges for the field.

Age-related macular degeneration (AMD) is the leading cause of vision loss in older adults and ul... more Age-related macular degeneration (AMD) is the leading cause of vision loss in older adults and ultimately leads to the death of photoreceptor cells in the macular area of the neural retina. Currently, treatments are only available for patients with the wet form of AMD. In this review, we describe recent approaches to develop cellbased therapies for the treatment of AMD. Recent research has focused on replacing the retinal pigment epithelium (RPE), a monolayer of cells vital to photoreceptor cell health. We discuss the various methods used to differentiate and purify RPE from human embryonic stem cells (HESC), and describe the surgical approaches being used to transplant these cells in existing and forthcoming clinical trials.

In the classical view of circadian clock organization, the daily rhythms of most organisms were t... more In the classical view of circadian clock organization, the daily rhythms of most organisms were thought to be regulated by a central, 'master' pacemaker, usually located within neural structures of the animal. However, with the results of experiments performed in zebrafish, mammalian cell lines and, more recently, mammalian tissues, this view has changed to one where clock organization is now seen as being highly decentralized. It is clear that clocks exist in the peripheral tissues of animals as diverse as Drosophila, zebrafish and mammals. In the case of Drosophila and zebrafish, these tissues are also directly lightresponsive. This light sensitivity and direct clock entrainability is also true for zebrafish cell lines and earlystage embryos. Using luminescent reporter cell lines containing clock gene promoters driving the expression of luciferase and single-cell imaging techniques, we have been able to show how each cell responds rapidly to a single light pulse by being shifted to a common phase, equivalent to the early day. This direct light sensitivity might be related to the requirement for light in these cells to activate the transcription of genes involved in DNA repair. It is also clear that the circadian clock in zebrafish regulates the timing of the cell cycle, demonstrating the wide impact that this light sensitivity and daily rhythmicity has on the biology of zebrafish.

The hypothalamic suprachiasmatic nuclei (SCN), the principal circadian oscillator in mammals, are... more The hypothalamic suprachiasmatic nuclei (SCN), the principal circadian oscillator in mammals, are synchronized to the solar day by the light-dark cycle, and in turn, they coordinate circadian oscillations in peripheral tissues. The tau mutation in the Syrian hamster is caused by a point mutation leading to a deficiency in the ability of Casein Kinase 1ε to phosphorylate its targets, including circadian PER proteins. How this accelerates circadian period in neural tissues is not known, nor is its impact on peripheral circadian oscillators established. We show that this mutation has no effect on per mRNA expression nor the nuclear accumulation of PER proteins in the SCN. It does, however, accelerate the clearance of PER proteins from the nucleus to an extent sufficient to explain the shortened circadian period of behavioral rhythms. The mutation also has novel, unanticipated consequences for circadian timing in the periphery, including tissue-specific phase advances and/or reduced amplitude of circadian gene expression. The results suggest that the tau mutation accelerates a specific phase, during mid-late subjective night of the SCN circadian feedback loop, rather than cause a global compression of the entire cycle. This reprogrammed output from the clock is associated with peripheral desynchrony, which in turn could account for impaired growth and metabolic efficiency of the mutant.

Zebrafish tissues and cell lines contain circadian clocks that respond directly to light 1,2 . Us... more Zebrafish tissues and cell lines contain circadian clocks that respond directly to light 1,2 . Using fluorescence-activated cell sorting, we have isolated clonal cell lines that contain the reporter construct, zfperiod4-luciferase 3 . Bioluminescent assays show that oscillations within cell populations are dampened in constant darkness. However, single-cell imaging reveals that individual cells continue to oscillate, but with widely distributed phases and marked stochastic fluctuations in free-running period. Because these cells are directly light responsive, we can easily follow phase shifts to single light pulses. Here we show that light acts to reset desynchronous cellular oscillations to a common phase, as well as stabilize the subsequent free-running period.

Molecular …, Jan 1, 2011

PurposeIn several species the retinal pigment epithelium (RPE) has the potential to transdifferen... more PurposeIn several species the retinal pigment epithelium (RPE) has the potential to transdifferentiate into retinal cells to regenerate functional retinal tissue after injury. However, this capacity for regeneration is lost in mammals. The synthetic retinoic acid derivative, fenretinide [N(4-hydroxyphenyl) retinamide], induces a neuronal-like phenotype in the human adult retinal pigment epithelial cell line (ARPE-19). These changes are characterized by the appearance of neural-like processes and the expression of neuronal markers not normally associated with RPE cells. Here we assess whether fenretinide can induce a neuroretinal cell phenotype in ARPE-19 cells, by examining retinal cell marker expression.MethodsARPE-19 cells were treated daily with culture medium containing either 3 μM fenretinide or dimethyl sulfoxide as a control for 7 days. Cells were processed for immunocytochemistry, western blotting, and for analysis by PCR to examine the expression of a panel of RPE, neural, and retinal-associated cellular markers, including classical and non-canonical opsins.ResultsTreatment with fenretinide for 7 days induced the formation of neuronal-like processes in ARPE-19 cells. Fenretinide induced the expression of the cone long wavelength sensitive opsin (OPN1lw) but not rhodopsin (RHO), while decreasing the expression of RPE cell markers. Many of the neuronal and retinal specific markers examined were expressed in both control and fenretinide treated cells, including those involved in photoreceptor cell development and the multipotency of neural retinal progenitor cells. Interestingly, ARPE-19 cells also expressed both photoreceptor specific and non-specific canonical opsins.ConclusionsThe expression of retinal-associated markers and loss of RPE cell markers in control ARPE-19 cells suggests that these cells might have dedifferentiated from an RPE cell phenotype under standard culture conditions. The expression of molecules, such as the transcription factors paired box 6 gene (PAX6), sex determining region Y-box 2 (SOX2), cone-rod homeobox (CRX), and neural retina leucine zipper (NRL), further implies that in culture these cells are predisposed toward a retinal progenitor-like state. The fenretinide-induced increase in photoreceptor cell markers, accompanied by a decrease in RPE cell markers, suggests that retinoids may play a role in the transdifferentiation of RPE cells. Importantly, our data show for the first time the expression of a vertebrate ciliary opsin (OPN1lw) and rhabdomeric-like opsin, opsin 4 (OPN4 also known as melanopsin) in a clonal cell line. Together these data suggest that ARPE-19 cells are primed for and possess the capacity to differentiate toward a retinal cell-like lineage.

… Ophthalmology & Visual …, Jan 1, 2011

Chronobiology …, Jan 1, 2006

Zebrafish are typically used as a model system to study various aspects of developmental biology,... more Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early-stage zebrafish embryos contain clocks that are directly light-responsive. We describe recent experiments using single-cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light-responsiveness of early-stage zebrafish embryos.

Molecular …, Jan 1, 2009

PurposeTo examine the ability of retinal pigment epithelial (RPE) cells derived from human embryo... more PurposeTo examine the ability of retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (HESC) to phagocytose photoreceptor outer segments, and to determine whether exposure to human retina induces any morphological changes in these cells.MethodsHESC-RPE cells were derived from a super-confluent preparation of the Shef1 HESC line. Pigmented colonies were isolated and expanded into pigmented monolayers on Matrigel™ matrix-coated dishes or filters. Cells were exposed to fluorescently labeled outer segments isolated from the porcine eye and assessed for phagocytic activity at regular intervals. Expression of molecules associated with RPE phagocytosis was analyzed by RT–PCR, immunocytochemistry, and western blot. The role of Mer Tyrosine Kinase (MERTK) in the phagocytosis of outer segments was investigated using antibodies directed against MERTK to block function. In a novel approach, cells were also exposed to fresh human neural retina tissue then examined by electron microscopy for evidence of phagocytosis and changes in cell morphology.ResultsHESC-derived RPE cells are capable of phagocytosing isolated porcine outer segments and express molecules associated with RPE-specific phagocytosis, including MERTK. Pre-incubation with antibodies against MERTK blocked phagocytosis of photoreceptor outer segments, but not polystyrene beads. HESC-RPE cells also phagocytosed outer segments in a novel human retinal explant system. Furthermore co-culture adjacent to human retina tissue in this preparation resulted in the appearance of features in HESC-derived RPE cells normally observed only as the RPE matures.ConclusionsThe ingestion of photoreceptor outer segments from an isolated population and an artificial ex vivo human retina system demonstrates HESC-derived RPE cells are functional. HESC-derived RPE possess the relevant molecules required for phagocytosis, including MERTK, which is essential for the phagocytosis of outer segments but not latex beads. Furthermore, some changes observed in cell morphology after co-culture with human retina may have implications for understanding the full development and differentiation of RPE cells.

The FASEB journal, Jan 1, 2003

Most mammals use changing annual daylength cycles to regulate pineal melatonin secretion and ther... more Most mammals use changing annual daylength cycles to regulate pineal melatonin secretion and thereby drive many physiological rhythms including reproduction, metabolism, immune function, and pelage. Prolonged exposure to short winter day lengths results in refractoriness, a spontaneous reversion to long-day physiological status. Despite its critical role in the timing of seasonal rhythms, refractoriness remains poorly understood. The aim of this study was therefore to describe cellular and molecular mechanisms driving the seasonal secretion of a key hormone, prolactin, in refractory Syrian hamsters. We used recently developed single cell hybridization and reporter assays to show that this process is initiated by timed reactivation of endocrine signaling from the pars tuberalis (PT) region of the pituitary gland, a well-defined melatonin target site, causing renewed activation of prolactin gene expression. This timed signaling is independent of per1 clock gene expression in the suprachiasmatic nuclei and PT and of melatonin secretion, which continue to track day length. Within the PT, there is also a continued short day-like profile of ICER expression, suggesting that the change in hormone secretion is independent of cAMP signaling. Our data thus identify the PT as a key anatomical structure involved in endogenous seasonal timing mechanisms, which breaks from prevailing day length-induced gene expression

Mechanisms of …, Jan 1, 2007

In this review we examine the potential of embryonic stem cells (ESCs) for use in the treatment o... more In this review we examine the potential of embryonic stem cells (ESCs) for use in the treatment of retinal diseases involving photoreceptors and retinal pigment epithelium (RPE). We outline the ontogenesis of target retinal cell types (RPE, rods and cones) and discuss how an understanding of developmental processes can inform our manipulation of ESCs in vitro. Due to their potential for cellular therapy, special emphasis is placed upon the derivation and culture of human embryonic stem cells (HESCs) and their differentiation towards a retinal phenotype. In terms of achieving this goal, we suggest that much of the success to date reflects permissive in vitro environments provided by established protocols for HESC derivation, propagation and neural differentiation. In addition, we summarise key factors that may be important for enhancing efficiency of retinal cell-type derivation from HESCs. The retina is an amenable component of the central nervous system (CNS) and as such, diseases of this structure provide a realistic target for the application of HESC-derived cellular therapy to the CNS. In order to further this goal, the second component of our review focuses on the cellular and molecular cues within retinal environments that may influence the survival and behaviour of transplanted cells. Our analysis considers both the potential barriers to transplant integration in the retina itself together with the remodelling in host visual centres that is known to accompany retinal dystrophy.

PLoS One, Jan 1, 2009

Transformation of somatic cells with a set of embryonic transcription factors produces cells with... more Transformation of somatic cells with a set of embryonic transcription factors produces cells with the pluripotent properties of embryonic stem cells (ESCs). These induced pluripotent stem (iPS) cells have the potential to differentiate into any cell type, making them a potential source from which to produce cells as a therapeutic platform for the treatment of a wide range of diseases. In many forms of human retinal disease, including age-related macular degeneration (AMD), the underlying pathogenesis resides within the support cells of the retina, the retinal pigment epithelium (RPE). As a monolayer of cells critical to photoreceptor function and survival, the RPE is an ideally accessible target for cellular therapy. Here we report the differentiation of human iPS cells into RPE. We found that differentiated iPS-RPE cells were morphologically similar to, and expressed numerous markers of developing and mature RPE cells. iPS-RPE are capable of phagocytosing photoreceptor material, in vitro and in vivo following transplantation into the Royal College of Surgeons (RCS) dystrophic rat. Our results demonstrate that iPS cells can be differentiated into functional iPS-RPE and that transplantation of these cells can facilitate the short-term maintenance of photoreceptors through phagocytosis of photoreceptor outer segments. Longterm visual function is maintained in this model of retinal disease even though the xenografted cells are eventually lost, suggesting a secondary protective host cellular response. These findings have identified an alternative source of replacement tissue for use in human retinal cellular therapies, and provide a new in vitro cellular model system in which to study RPE diseases affecting human patients.

Current biology, Jan 1, 2003

exhibited significantly reduced paired testes weight ( ) and 24 hr plasma prolactin concentration... more exhibited significantly reduced paired testes weight ( ) and 24 hr plasma prolactin concentration ( ) compared to the LD group. However, following exposure to SD for 28 weeks (SD-R group), the paired testes weight and plasma prolactin concentration were significantly elevated back to LD-like values. There was no significant daily rhythm of prolactin over the 24 hr Oxford Road Manchester M13 9P sampling period for any of the groups ( ). Pineal melatonin concentration was elevated between zeit-United Kingdom geber time (ZT) 20 and 24 in LD and between ZT14 and ZT22 in both SD groups ( ). This finding is consistent with previous studies in this species [5, 6] Summary and indicates refractoriness of the reproductive and neuroendocrine axis to the SD photoperiod and prevail-In many seasonally breeding rodents, reproduction and metabolism are activated by long summer days ing SD melatonin profile.

Experimental …, Jan 1, 2008

Nature cell biology, Jan 1, 2005