Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes, mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture, hES cells expressing cell-surface NGFR protein (CD271, p75NTR) were isolated by immunoaffinity adsorption, and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression, examined by quantitative RT-PCR, found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR, SNAI1, NTRK3, SOX9, and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer, mRNAs typifying adult stromal stem cells were detected, including BMI1, KIT, NES, NOTCH1, and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1, B3GNT7, PTDGS, and ALDH3A1 were upregulated. mRNA for keratocan (KERA), a cornea-specific proteoglycan, was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate, a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells, therefore, may provide a renewable source of material for development of treatment of corneal stromal opacities.

Following herpes simplex virus type 1 (HSV-1) ocular infection of C57BL/6 mice, activated CD8؉ T cells specific for an immunodominant epitope on HSV-1 glycoprotein B (gB-CD8 cells) establish a stable memory population in HSV-1 latently infected trigeminal ganglia (TG), whereas non-HSV-specific CD8 ؉ T cells are lost over time. The retention and activation of gB-CD8 cells appear to be influenced by persistent viral antigenic exposure within the latently infected TG. We hypothesized that the low-level expression of gB from its native promoter before viral DNA synthesis is critical for the retention and activation of gB-CD8 cells in the TG during HSV-1 latency and for their ability to block HSV-1 reactivation from latency. To test this, we created a recombinant HSV-1 in which gB is expressed only after viral DNA synthesis from the true late gC promoter (gCp-gB). Despite minor growth differences compared to its rescuant in infected corneas, gCp-gB was significantly growth impaired in the TG and produced a reduced latent genome load. The gCp-gB-and rescuantinfected mice mounted similar gB-CD8 effector responses, but the size and activation phenotypes of the memory gB-CD8 cells were diminished in gCp-gB latently infected TG, suggesting that the stimulation of gB-CD8 cells requires gB expression before viral DNA synthesis. Surprisingly, late gB expression did not compromise the capacity of gB-CD8 cells to inhibit HSV-1 reactivation from latency in ex vivo TG cultures, suggesting that gB-CD8 cells can block HSV-1 reactivation at a very late stage in the viral life cycle. These data have implications for designing better immunogens for vaccines to prevent HSV-1 reactivation.Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen that is responsible for repeated corneal infections that can induce blinding keratitis. The murine model of ocular HSV-1 infection has elucidated the role of host immunity in the establishment and maintenance of viral latency in trigeminal ganglia (TG). HSV-1 infection of a scarified mouse cornea leads to a short-lived epithelial lesion caused by acute viral replication in and destruction of corneal epithelial cells. During replication in culture, viral genes are expressed in a tightly regulated temporal cascade characterized by the sequential expression of immediate-early (IE) (␣) genes and early (␤) genes before viral DNA synthesis. The late ␥ genes are maximally expressed after viral DNA replication and can be subdivided into ␥1 genes, which are expressed in small amounts before viral DNA replication, and ␥2 genes, which are absolutely dependent on DNA replication for expression (7). Confirmation of the expression kinetics of HSV-1 genes in vivo has proven to be difficult, as uniform infections cannot be established. Previous studies seeking to address viral replication kinetics in neurons have given rise to controversial conclusions (31). However, our group has previously demonstrated that during reactivation, the ␥1 gene promoter of glycoprotein B (gB) is active before the ␥2 gene...

S U M M A R YImmunostaining in transgenic mice carrying the lac z gene can be used to map gene and protein distribution in a single tissue. In this study, we examined inner ears from ephrin-B3 homozygous and ephrin-B2 heterozygous mice. Ephrin-B3 lac z expression was limited in these mice. However, immunostaining revealed ephrin-B3 throughout cochlear and vestibular regions. Immunoreactivity was absent in ephrin-B3-homozygous null mutants, demonstrating the specificity of the antibody. Ephrin-B2 lac z reactivity was detected in a limited number of cells in cochlear and vestibular regions. Different immunostaining patterns were found with different antibodies. Comparison with lac z expression indicated which antibody was specific for the transmembrane-bound ephrin-B2 ligand.

Rift Valley fever virus (RVFV) infection leads to eye damage in humans in up to 10% of reported cases. Permanent blindness occurs in 50% of individuals with significant retinal scarring.