Peter J. Coffey

Protective Effects of Human iPS-Derived Retinal Pigment Epithelium Cell Transplantation in the Retinal Dystrophic Rat

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. Long-term 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.

Face processing impairments after encephalitis: amygdala damage and recognition of fear

Face processing and facial emotion recognition were investigated in five post-encephalitic people of average or above-average intelligence. Four of these people (JC, YW, RB and SE) had extensive damage in the region of the amygdala. A fifth post-encephalitic person with predominantly hippocampal damage and relative sparing of the amygdala (RS) participated, allowing us to contrast the effects of temporal lobe damage including and excluding the amygdala region. The findings showed impaired recognition of fear following bilateral temporal lobe damage when this included the amygdala. For JC, this was part of a constellation of deficits on face processing tasks, with impaired recognition of several emotions. SE, YW and RB, however, showed relatively circumscribed deficits. Although they all had some problems in recognizing or naming famous faces, and had poor memory for faces on the Warrington Recognition Memory Test, none showed a significant impairment on the Benton Test of Facial Recognition, indicating relatively good perception of the faces physical structure. In a test of recognition of basic emotions (happiness, surprise, fear, sadness, disgust and anger), SE, YW and RB achieved normal levels of performance in comparison to our control group for all emotions except fear. Their results contrast with those of RS, with relative sparing of the amygdala region and unimpaired recognition of emotion, pointing clearly toward the importance of the amygdala in the recognition of fear.

Elucidating the phenomenon of HESC-derived RPE: Anatomy of cell genesis, expansion and retinal transplantation

Healthy Retinal Pigment Epithelium (RPE) cells are required for proper visual function and the phenomenon of RPE derivation from Human Embryonic Stem Cells (HESC) holds great potential for the treatment of retinal diseases. However, little is known about formation, expansion and expression profile of RPE-like cells derived from HESC (HESC-RPE). By studying the genesis of pigmented foci we identified OTX1/2-positive cell types as potential HESC-RPE precursors. When pigmented foci were excised from culture, HESC-RPE expanded to form extensive monolayers, with pigmented cells at the leading edge assuming a precursor role: de-pigmenting, proliferating, expressing keratin 8 and subsequently re-differentiating. As they expanded and differentiated in vitro, HESC-RPE expressed markers of both developing and mature RPE cells which included OTX1/2, Pax6, PMEL17 and at low levels, RPE65. In vitro, without signals from a developing retinal environment, HESC-RPE could produce regular, polarised monolayers with developmentally important apical and basal features. Following transplantation of HESC-RPE into the degenerating retinal environment of Royal College of Surgeons (RCS) dystrophic rats, the cells survived in the subretinal space, where they maintained low levels of RPE65 expression and remained out of the cell cycle. The HESC-RPE cells responded to the in vivo environment by downregulating Pax6, while maintaining expression of other markers. The presence of rhodopsin-positive material within grafted HESC-RPE indicates that in the future, homogenous transplants of this cell type may be capable of supporting visual function following retinal dystrophy.

Subretinal transplantation of genetically modified human cell lines attenuates loss of visual function in dystrophic rats

Royal College of Surgeons rats are genetically predisposed to undergo significant visual loss caused by a primary dysfunction of retinal pigment epithelial (RPE) cells. By using this model, we have examined the efficacy of subretinal transplantation of two independent human RPE cell lines each exhibiting genetic modifications that confer long-term stability in vitro. The two cell lines, a spontaneously derived cell line (ARPE19) and an extensively characterized genetically engineered human RPE cell line (h1RPE7), which expresses SV40 large T (tumor) antigen, were evaluated separately. Both lines result in a significant preservation of visual function as assessed by either behavioral or physiological techniques. This attenuation of visual loss correlates with photoreceptor survival and the presence of donor cells in the areas of rescued photoreceptors at 5 months postgrafting (6 months of age). These results demonstrate the potential of genetically modified human RPE cells for ultimate application in therapeutic transplantation strategies for retinal degenerative diseases caused by RPE dysfunction.

RPE transplantation and its role in retinal disease.

Retinal pigment epithelial (RPE) transplantation aims to restore the subretinal anatomy and re-establish the critical interaction between the RPE and the photoreceptor, which is fundamental to sight. The field has developed over the past 20 years with advances coming from a large body of animal work and more recently a considerable number of human trials. Enormous progress has been made with the potential for at least partial restoration of visual function in both animal and human clinical work. Diseases that have been treated with RPE transplantation demonstrating partial reversal of vision loss include primary RPE dystrophies such as the merTK dystrophy in the Royal College of Surgeons (RCS) rat and in humans, photoreceptor dystrophies as well as complex retinal diseases such as atrophic and neovascular age-related macular degeneration (AMD). Unfortunately, in the human trials the visual recovery has been limited at best and full visual recovery has not been demonstrated. Autologous full-thickness transplants have been used most commonly and effectively in human disease but the search for a cell source to replace autologous RPE such as embryonic stem cells, marrow-derived stem cells, umbilical cord-derived cells as well as immortalised cell lines continues. The combination of cell transplantation with other modalities of treatment such as gene transfer remains an exciting future prospect. RPE transplantation has already been shown to be capable of restoring the subretinal anatomy and improving photoreceptor function in a variety of retinal diseases. In the near future, refinements of current techniques are likely to allow RPE transplantation to enter the mainstream of retinal therapy at a time when the treatment of previously blinding retinal diseases is finally becoming a reality.

Long-term preservation of cortically dependent visual function in RCS rats by transplantation.

Cell transplantation is one way of limiting the progress of retinal degeneration in animal models of blinding diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Here we transplanted a human retinal pigment epithelial (RPE) cell line into the subretinal space of one such model, the Royal College of Surgeons (RCS) rat, and showed, using head tracking to moving stripes and pattern discrimination in conjunction with single-unit cortical physiology, that cortically mediated vision can be preserved with this treatment.

Cell transplantation as a treatment for retinal disease.

It has been shown that photoreceptor degeneration can be limited in experimental animals by transplantation of fresh RPE to the subretinal space. There is also evidence that retinal cell transplants can be used to reconstruct retinal circuitry in dystrophic animals. Here we describe and review recent developments that highlight the necessary steps that should be taken prior to embarking on clinical trials in humans.

Stem cells in retinal regeneration: past, present and future

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, Stargardts 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.

Spectroscopic analysis of neural activity in brain: increased oxygen consumption following activation of barrel cortex.

This research investigates the hemodynamic response to stimulation of the barrel cortex in anaesthetized rats using optical imaging and spectroscopy (Bonhoeffer and Grinvald, 1996; Malonek and Grinvald, 1996; Mayhew et al., 1999). A slit spectrograph was used to collect spectral image data sequences. These were analyzed using an algorithm that corrects for the wavelength dependency in the optical path lengths produced by the light scattering properties of tissue. The analysis produced the changes in the oxy- and deoxygenation of hemoglobin following stimulation. Two methods of stimulation were used. One method mechanically vibrated a single whisker, the other electrically stimulated the whisker pad. The electrical stimulation intensity varied from 0.4 to 1.6 mA. The hemodynamic responses to stimulation increased as a function of intensity. At 0.4 mA they were commensurate with those from the mechanical stimulation; however, the responses at the higher levels were greater by a factor of approximately 10. For both methods of data collection, the results of the spectroscopic analysis showed an early increase in deoxygenated hemoglobin (Hbr) with no evidence for a corresponding decrease in oxygenated hemoglobin (HbO(2)). Evidence for increased oxygen consumption (CMRO(2)) was obtained by converting the fractional changes in blood volume (Hbt) into estimates of changes in blood flow (Grubb et al., 1974) and using the resulting time course to scale the fractional changes in Hbr. The results show an early increase CMRO(2) peaking approximately 2 s after stimulation onset. Using these methods, we find evidence for increased oxygen consumption following increased neural activity even at low levels of stimulation intensity.

Complement factor H deficiency in aged mice causes retinal abnormalities and visual dysfunction.

Age-related macular degeneration is the most common form of legal blindness in westernized societies, and polymorphisms in the gene encoding complement factor H (CFH) are associated with susceptibility to age-related macular degeneration in more than half of affected individuals. To investigate the relationship between complement factor H (CFH) and retinal disease, we performed functional and anatomical analysis in 2-year-old CFH-deficient (cfh−/−) mice. cfh−/− animals exhibited significantly reduced visual acuity and rod response amplitudes on electroretinography compared with age-matched controls. Retinal imaging by confocal scanning laser ophthalmoscopy revealed an increase in autofluorescent subretinal deposits in the cfh−/− mice, whereas the fundus and vasculature appeared normal. Examination of tissue sections showed an accumulation of complement C3 in the neural retina of the cfh−/− mice, together with a decrease in electron-dense material, thinning of Bruchs membrane, changes in the cellular distribution of retinal pigment epithelial cell organelles, and disorganization of rod photoreceptor outer segments. Collectively, these data show that, in the absence of any specific exogenous challenge to the innate immune system, CFH is critically required for the long-term functional health of the retina.