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.

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.

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

BACKGROUND: CD133 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. METHODS: Retinal 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. RESULTS: We 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. CONCLUSION: These 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.

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 10(5) 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 10(5) 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.

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-gamma (LPS/IFNgamma) 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-IFNgamma 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/IFNgamma-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/IFNgamma stimulation, their migration is sustained via CD200R stimulation maintaining their potential to migrate in response to injury.