Da Carter

Neural progenitor cells from postmortem adult human retina

Background: Given the presence of neural progenitor cells (NPC) in the retina of other species capable of differentiating into multiple neural components, the authors report the presence of NPC in the adult human retina. A resident population of NPC suggests that the retina may constitutively replace neurons, photoreceptors, and glia. Methods: Adult human postmortem retinal explants and cell suspensions were used to generate cells in tissue culture that display the features of NPC. The phenotype of cells and differentiation into neurons was determined by immunocytochemistry. Dividing cells were labelled with 5-bromo-2-deoxyuridine (BrdU) and neurospheres were generated and passaged. Results: Cells labelled with nestin, neurofilament M (NFM), rhodopsin, or glial fibrillary acidic protein (GFAP) grew out from explant cultures. BrdU labelling of these cells occurred only with basic fibroblast growth factor (FGF-2). Dissociated retina and pars plana generated primary neurospheres. From primary neurospheres, NPC were passaged to generate secondary neurospheres, neurons, photoreceptors, and glia. BrdU labelling identified dividing cells from neurospheres that differentiated to express NFM and rhodopsin. Conclusion: The adult human retina contains NPC and may have the potential to replace neurons and photoreceptors. This has implications for the pathogenesis and treatment of retinal disorders and degenerations, including glaucoma, and those disorders associated with retinal scarring.

Control of myeloid activity during retinal inflammation

Combating myeloid cell‐mediated destruction of the retina during inflammation or neurodegeneration is dependent on the integrity of homeostatic mechanisms within the tissue that may suppress T cell activation and their subsequent cytokine responses, modulate infiltrating macrophage activation, and facilitate healthy tissue repair. Success is dependent on response of the resident myeloid‐cell populations [microglia (MG)] to activation signals, commonly cytokines, and the control of infiltrating macrophage activation during inflammation, both of which appear highly programmed in normal and inflamed retina. The evidence that tissue CD200 constitutively provides down‐regulatory signals to myeloid‐derived cells via cognate CD200‐CD200 receptor (R) interaction supports inherent tissue control of myeloid cell activation. In the retina, there is extensive neuronal and endothelial expression of CD200. Retinal MG in CD200 knockout mice display normal morphology but unlike the wild‐type mice, are present in increased numbers and express nitric oxide synthase 2, a macrophage activation marker, inferring that loss of CD200 or absent CD200R ligation results in “classical” activation of myeloid cells. Thus, when mice lack CD200, they show increased susceptibility to and accelerated onset of tissue‐specific autoimmunity.

Nestin positive cells in adult human retina and in epiretinal membranes

Background/aim: Nestin is an intermediate filament marker for neural progenitor cells. The authors aimed to identify nestin positive cells in adult human retina and within surgically removed epiretinal membranes. Methods: Adult human retina and epiretinal membranes were studied. Tissue was fixed and processed for semithin sections or whole mount preparations for immunohistochemical detection of nestin and glial fibrillary acidic protein (GFAP) expression. Results: Nestin positive cells are most prominent at the ora serrata, possess fibrillary processes, small amounts of perinuclear cytoplasm, and are arranged radially within or superficially on the retina. In the posterior retina, speckled cytoplasmic nestin staining is seen around the nuclei of neurons. In the peripapillary retina most of the cells in the retinal ganglion cell layer are nestin positive. These cells appear to represent nestin positive neurons. Speckled cells are also seen in the myelinated portion of the optic nerve. In epiretinal membranes patches of elongated nestin positive cells were found. These cells were also positive for GFAP. Conclusions: Some neurons and glia in the adult human retina are nestin positive. Their pattern in anterior retina suggests an analogy with the ciliary marginal zone found in many other species. The role of these cells in pathological responses to retinal disease is suggested by the presence of large numbers of ectopic nestin positive cells in epiretinal membranes. The authors hypothesise that nestin positive cells represent a population of progenitor cells from normal adult human retina that differentiate to make up retinal scar tissue.

CD200 maintains microglial potential to migrate in adult human retinal explant model.

Purpose. Retinal microglia (MG) migrate in response to injury, degeneration and inflammation dependent upon both soluble and cognate signals they receive. Previously we found that lipopolysaccharide/interferon-γ (LPS/IFNγ) stimulation induces a paradoxical IL-10 mediated suppression of MG migration from retinal explants. Given the high expression of neuronal CD200, which can induce down regulation of CD200 receptor-positive MG activation and neuronal fractalkine expression potentially stimulating MG migration, we wished to further examine their respective roles in the maintenance of MG activation and migration. Methods. A human retinal explant model of MG migration was used. CD200 receptor and fractalkine receptor stimulation was achieved by addition to explants of CD200:Fc fusion protein and recombinant cytokine respectively, with or without LPS-IFNγ stimulation that is known to suppress migration. Cell migration and cell activation (iNOS expression) was counted and assessed by numbers of CD45+ cells by immunofluorescence and standardised flow cytometric bead array analysis was performed for cytokine production. Results. Retinal explants expressed fractalkine and CX3CR1 immunohistochemically and by PCR. Addition of Fractalkine and not CD200:Fc induced MG migration from retinal explants. However LPS/IFNγ-induced suppression of MG migration could only be restored in the presence of CD200:Fc, whilst MG remained iNOS-negative and generated IL-10. Conclusions. Microglial responses are tightly governed within retina. Although MG do not classically activate following LPS/IFNγ stimulation, their migration is sustained via CD200R stimulation maintaining their potential to migrate in response to injury.

CD133+ adult human retinal cells remain undifferentiated in Leukaemia Inhibitory Factor (LIF).

BackgroundCD133 is a cell surface marker of haematopoietic stem and progenitor cells. Leukaemia inhibitory factor (LIF), sustains proliferation and not differentiation of embryonic stem cells. We used CD133 to purify adult human retinal cells and aimed to determine what effect LIF had on these cultures and whether they still had the ability to generate neurospheres.MethodsRetinal cell suspensions were derived from adult human post-mortem tissue with ethical approval. With magnetic automated cell sorting (MACS) CD133+ retinal cells were enriched from post mortem adult human retina. CD133+ retinal cell phenotype was analysed by flow cytometry and cultured cells were observed for proliferative capacity, neuropshere generation and differentiation with or without LIF supplementation.ResultsWe demonstrated purification (to 95%) of CD133+ cells from adult human postmortem retina. Proliferating cells were identified through BrdU incorporation and expression of the proliferation markers Ki67 and Cyclin D1. CD133+ retinal cells differentiated whilst forming neurospheres containing appropriate lineage markers including glia, neurons and photoreceptors. LIF maintained CD133+ retinal cells in a proliferative and relatively undifferentiated state (Ki67, Cyclin D1 expression) without significant neurosphere generation. Differentiation whilst forming neurospheres was re-established on LIF withdrawal.ConclusionThese data support the evidence that CD133 expression characterises a population of cells within the resident adult human retina which have progenitor cell properties and that their turnover and differentiation is influenced by LIF. This may explain differences in retinal responses observed following disease or injury.

Lipopolysaccharide/interferon-γ and not transforming growth factor β inhibits retinal microglial migration from retinal explant

Background/aims: The retina possesses a rich network of CD45+ positive myeloid derived cells that both surround inner retinal vessels and lie within the retina (microglia). Microglia migrate and accumulate in response to neurodegeneration and inflammation. Although microglia express MHC class II, their role remains undefined. The aims of this study are to investigate changes in human microglia phenotype, migration, and activation status in response to pro-inflammatory and anti-inflammatory stimulation. Methods: Donor eyes were obtained from the Bristol Eye Bank with consent and whole retina was removed. 5 mm retinal trephines were cultured in glucose enhanced RPMI on cell culture insert membranes for up to 72 hours. The effects of lipopolysaccharide/interferon-γ (LPS/IFNγ) and transforming growth factor β inhibits (TGFβ) stimulation, alone or in combination, on migration, phenotype, and activation status (iNOS expression) of microglia were studied using immunofluorescence and cytokine analysis by ELISA. Results: CD45+ MHC class II+ retinal microglia were observed within retinal explants, and in culture microglia readily migrated, adhered to culture membrane, downregulated MHC class II expression, and produced interleukin 12 (IL-12) and tumour necrosis factor α (TNFα). Following LPS/IFNγ stimulation microglia remained MHC class II− iNOS−, and secreted IL-10. Migration was suppressed and this could be reversed by neutralising IL-10 activity. TGFβ did not affect ability of microglia to migrate and was unable to reverse LPS/IFNγ induced suppression. Conclusions: Microglia readily migrate from retinal explants and are subsequently MHC class II−, iNOS−, and generate IL-12. In response to LPS/IFNγ microglia produce IL-10, which inhibits both their migration and activation. TGFβ was unable to counter LPS/IFNγ effects. The data infer that microglia respond coordinately, dependent upon initial cytokine stimulation, but paradoxically respond to classic myeloid activation signals.

Microglia derived IL-6 suppresses neurosphere generation from adult human retinal cell suspensions

Following retinal degeneration or inflammation that disrupts tissue architecture, there is limited evidence of tissue regeneration, despite evidence of cells with progenitor properties in the adult human retina at all ages. With the prospect of tissue/cell transplantation, redressing homeostasis whilst overcoming glial barrier or gliosis remains key to successful graft versus host integration and functional recovery. Activated human retinal microglia (MG) secrete cytokines, including IL-6, which may suppress neurogenesis or cellular (photoreceptor) replacement. To investigate this hypothesis, adult human retinal explants were cultured in cytokine-conditioned media (TNFalpha, TGFbeta, LPS/IFNgamma) to activate microglia in situ. Following culture of retinal explants for 4 days, supernatant conditioned by resulting migrated microglia was collected after a further 3 days and fed to retinal cell suspensions (RCS). Neurosphere (NS) generation and survival analysis was performed after 7 and 14 days in culture, with or without addition of conditioned media and with or without concomitant IL-6 neutralisation. Neurosphere phenotype was analysed by immunohistochemistry and cell morphology. Migratory MG from retinal explants were activated (iNOS-positive) and expressed CD45, CD11b, and CD11c. LPS/IFNgamma-activated MG conditioned media (MG-CM) contained significant levels of IL-6 (1265 +/- 143) pg/ml, which inhibited neurosphere generation within RCS in the presence of optimal neurosphere generating N2-FGF2 culture medium. Neutralising IL-6 activity reinstated NS generation and the differentiation capacity was maintained in the spheres that formed. Even in the presence of high levels of IL-6, those few NS that did form demonstrated a capacity to differentiate. The data supports activated MG-derived IL-6 influence retinal cell turnover.

The Effect of Postmortem Time, Donor Age and Sex on the Generation of Neurospheres from Adult Human Retina.

Background/Aim: Postmortem adult human retina contains pluripotent progenitor cells capable of forming neurospheres with different retinal cell types. The authors examine whether this is the case at all ages and at different postmortem times. Methods: Adult human postmortem retina-derived cell suspensions generated neurospheres in fibroblast growth factor 2 and N2 supplement. The yield of neurospheres from limited dilution or single cell cultures is very low so the authors studied cells generated per 105 viable cells from a cell suspension derived from whole retina. Retinal tissue from donors aged 18–91 at various postmortem times (between 23–44 h) was studied in the context of generation rate and time for neurospheres. Results: The potential to generate neurospheres from adult human retina remains throughout life. Neurosphere cellular components were not affected by donor age or postmortem time (they contained nestin+, glial fibrillary acidic protein+ and neurofilament+ cells). An average of 34.36 neurospheres were generated per 105 viable cells. After a few days in culture neurospheres begin to form. The time for this to occur was independent of donor age but prolonged at longer postmortem times. No significant effect of donor sex was found. Conclusion: Neurosphere-forming retinal progenitor cells are found in adult human retina throughout life. This cell population is a potential target for therapeutic intervention to influence repair and regeneration of the retina.

Cytokines and immunopathogenesis of intraocular posterior segment inflammation

Summary Intraocular inflammatory eye disease remains a significant cause of visual handicap in the working age population within developed countries. The development of inflammation is unquestionably multifactorial, including genetic susceptibility, nutritional status, innate stimuli such as injury and concomitant ocular and systemic infection, and presence of autoreactive T cells. When no overt infectious agent is identified in uveitic patients, we presume that autoimmunity is the underlying pathogenesis. Although retinal antigen-specific Th1 CD4 + T cells mediate experimental models of posterior uveitis, confirmation of such in man has yet to be fully established. Antigen is the initiator of all acquired immune responses, whereupon the primary function of the immune system is to recognise exogenous antigens and eliminate pathogens. Additionally, the maintenance of immune regulation is brought about by tolerance to self-antigens acquired during development (central tolerance) or actively during adult life (peripheral tolerance) and in the case of exogenous antigens (viral and bacterial) immune responses are, in part, kept in check by inhibitory cytokines and soluble cytokine receptors. The dynamic and continual regulation of immune responses is orchestrated by cytokines, controlling leukocyte behaviour and activation including antigen-presenting cells (dendritic cells; DC), antigen-specific T cells, and non-specific leukocytes such as macrophage and granulocytes. Moreover, tissues such as the eye are endowed with powerful immunomodulatory mechanisms, in which cytokines play a pivotal role to control immune responses, limit tissue damage, and restore homoeostasis.

Functional analysis of retinal microglia and their effects on progenitors.

The identification of stem/progenitor cells within the retinal neural environment has opened up the possibility of therapy via cellular replacement and/or reprogramming of resident cell populations. Within the neuro-retinal niche, following injury or in disease states (including inflammation and degeneration), cellular responses affect tissue homeostasis, reduce cell density, disrupt tissue architecture, and produce scar formation. Microglia (resident retinal immune cell tissue macrophage) are key to the maintenance of retinal homeostasis and are implicated in responses that may influence the control and behavior of retinal progenitors. Factors to consider in the generation of a transplantable cell resource with good migratory and integrative capacity include their yield, purity, and functional viability. Utilizing human postmortem retina, we have created a research platform to isolate, culture, and characterize adult retinal microglia as well as analyze their effect on retinal progenitors. Here, we describe techniques using magnetic labeled bead cell separation to isolate pure populations of retinal CD133(+) precursor cells and CD11b(+) microglia from primary adult retinal cell suspensions (RCSs), enabling flow cytometric cell phenotypic and qPCR genotypic analysis, as well as functional analysis by real-time ratiometric calcium imaging.