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Loren Alexis Ornelas, Yelena Bykhovskaya, Dhruv Sareen, Yaron S Rabinowitz; Derivation and Characterization of Human Induced Pluripotent Stem Cells from Stromal Keratocytes of Patients with Keratoconus. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4201.
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Keratoconus (KC) is a frequent cause of visual abnormalities due to corneal thinning and loss of function. It causes a significant health burden due to donor organ shortage and high cost. It is characterized by a complex inheritance of multiple genetic defects with several gene candidates (Lu et al., 2012). Disease progression is poorly understood due to a lack of cellular and animal models. A new paradigm in disease modeling is the creation of human induced pluripotent stem cells (hiPSCs). We took advantage of the fact that hiPSCs can maintain residual epigenetic memory of the original cell that they are derived from. Here we show corneal keratocyte-derived hiPSCs from KC patients and healthy individuals.
Primary keratocytes were isolated from freshly isolated corneas by collagenase digestion at 37oC, rinsed in keratocyte media (DMEM/F12, N2, B27, bFGF) and plated. To induced proliferation, isolated keratocytes were then “activated” by addition of serum and reprogramming was performed using episomal plasmid transfection expressing pluripotency factors. Cultures were plated on Matrigel and fed a non-serum containing medium supplemented with reprogramming small molecules for 18 days. Colonies resembling PSC morphology were isolated and plated on Matrigel coated plates. To induce re-differentiation to keratocytes, hiPSCs from a GFP expressing reporter line were plated on isolated Descemet’s membrane using 12 well Cell Crown.
Human IPSCs were successfully generated from primary keratocytes isolated from fresh human corneas of healthy individuals and KC patients after serum activation. Twelve clones were selected for further expansion and 3 clones were characterized using battery of pluripotency assays. Further, we demonstrated that iPSCs expanded and survived during differentiation in keratocyte conditioned medium after direct plating on Descemet’s membrane isolated from donor corneas.
In this study we demonstrate for the first time efficient generation of KC-specific human iPSCs from stromal keratocytes. These keratocyte-derived iPSCs exhibited all properties of human pluripotent stem cells, including morphology and expression of genes and surface markers indicative of pluripotency. These cells will provide a valuable “disease-in-a-dish” model to study KC pathogenesis and progression, and aid in the development of novel therapies.
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