Differentiation of Human Embryonic Stem Cells into Cells with Corneal Keratocyte Phenotype

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. © 2013 Chan et al

Cone photoreceptor macular function and recovery after photostress in early non-exudative age-related macular degeneration

John D Rodriguez,1 Keith Lane,1 David A Hollander,1,2 Aron Shapiro,1 Sunita Saigal,1 Andrew J Hertsenberg,1 Garrick Wallstrom,3 Divya Narayanan,1 Endri Angjeli,1 Mark B Abelson1,4 1Ora, Inc., Andover, MA, USA; 2Jules Stein Eye Institute, University of California, Los Angeles, CA, USA; 3Statistics and Data Corporation, Tempe, AZ, USA; 4Department of Ophthalmology, Harvard Medical School, Boston, MA, USA Purpose: To identify parameters from cone function and recovery after photostress that detect functional deficits in early non-exudative age-related macular degeneration (AMD) and to determine the repeatability of these parameters. Methods: Cone-mediated visual function recovery after photostress was examined in three groups of subjects: young normal subjects (ages 20–29; N=8), older normal subjects (ages 50–90; N=9), and early non-exudative AMD subjects (ages 50–90; N=12). Eight AMD and four normal subjects were retested 1 year after the initial evaluation. Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity (VA) and parameters of cone function (baseline cone sensitivity and cone recovery half-life following photobleach) were measured and compared between AMD and normal subjects. Short-term repeatability was assessed for each subject’s initial evaluation. Long-term repeatability was assessed by comparing outcomes from the initial evaluation and 1-year follow-up. Results: The mean baseline cone threshold was significantly worse in subjects with early AMD compared to older normal subjects (-1.80±0.04 vs -1.57±0.06 log cd/m2 p=0.0027). Moreover, the baseline cone threshold parameter exhibited good short-term (intraclass correlation coefficient [ICC]=0.88) and long-term (ICC=0.85) repeatability in all subjects. The cone intercept parameter and ETDRS VA were not significantly different between AMD and older normal subject groups. Cone recovery half-life was significantly different between older normal and AMD subject groups (p=0.041). Neither ETDRS VA nor cone function parameters were significantly different for any group at the 1-year follow-up. Conclusion: The baseline cone threshold shows potential as a novel parameter to assess visual dysfunction in early AMD. This outcome consistently detected deficits in AMD subjects, and differentiated them from age-matched controls with high test–retest repeatability. Keywords: AMD, photobleach, photoreceptor, cone recovery, Ora LU