title: Using induced pluripotent stem cell-derived conditional medium to attenuate the light-induced photodamaged retina of rats.
abstract: BACKGROUND
  Light injury to photoreceptor cells and retinal pigment epithelium may lead to oxidative stress and irreversible degeneration of retina, especially degeneration of the high energy-demanded macula. The model of retinal photodamage could be applied to age-related macular degeneration and other degenerative retinal diseases for exploring new treatments. Based on broadly investigated induced pluripotent stem cells (iPSC) in the field of retinal degeneration, we aimed to clarify further how the interaction progresses between iPSC-conditional medium (CM) and light-damaged retina.
  
  
  METHODS
  iPSCs were generated from murine embryonic fibroblasts of C57/B6 mice by retroviral transfection of three factors: Oct4, Sox2, and Klf4. Cytokine array was performed to analyze the components of CM. Sprague-Dawley rats receiving white light exposure to retina were viewed as an animal model of light injury. The rats were divided into four subgroups: light-injured rats receiving intravitreal injection of iPSC-CM, apoptotic iPSC-CM, or sodium phosphate buffer (PBS); and a control group without light damage. The electroretinography and thickness of outer nuclear layer were measured to document the therapeutic effects in each condition. Apoptosis arrays for detecting annexin V and caspase 3 were performed in the retinal tissues from each group.
  
  
  RESULTS
  Murine embryonic fibroblasts were induced into iPSCs and expressed the marker genes similar to embryonic stem cells. These iPSCs can differentiate into Embryoid bodies (EBs), three germ layers in vitro and develop teratoma in severe combined immunodeficiency mice. The quantitative polymerase chain reaction of our iPSC-CM showed significantly elevated fibroblast growth factor-2, glial cell-derived neurotrophic factor, and insulin-like growth factor-binding proteins-1, -2, and -3. Compared to rats without photodamage, the light-injured rats receiving iPSC-CM had less reduction of outer nuclear layer thickness on Day 21 than other groups treated with either PBS or apoptotic iPSC-CM. In the same animal model, both a- and b-waves of electroretinography measurement in the group treated with iPSC-CM were significantly maintained compared to the control group and others with apoptotic iPSC-CM or PBS treatment. The apoptosis assay also demonstrated lower levels of annexin V and caspase 3 in the group with iPSC-CM treatment than in other groups presenting increasing apoptotic markers.
  
  
  CONCLUSIONS
  The conditional medium of iPSCs contains plenty of cytoprotective, immune-modulative and rescue chemicals, contributing to the maintenance of neuronal function and retinal layers in light-damaged retina compared with apoptotic iPSC-CM and PBS. The antiapoptotic effect of iPSC-CM also shows promise in restoring damaged neurons. This result demonstrates that iPSC-CM may serve as an alternative to cell therapy alone to treat retinal light damage and maintain functional and structural integrity of the retina.
authors.affiliation: Department of Optics and Photonics, National Central University, Chung-Li, Taiwan, ROC; Bade Veterans Home, Taoyuan, Taiwan, ROC. Electronic address: huamingchang@gmail.com.
authors.forename: Hua-Ming
authors.initials: HM
authors.lastname: Chang
authors.affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan, ROC; Division of Ophthalmology, National Yang-Ming University Hospital, I-Lan, Taiwan, ROC.
authors.forename: Kuo-Hsuan
authors.initials: KH
authors.lastname: Hung
authors.affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan, ROC; Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan, ROC.
authors.forename: Chih-Chien
authors.initials: CC
authors.lastname: Hsu
authors.affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan, ROC; Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan, ROC.
authors.forename: Tai-Chi
authors.initials: TC
authors.lastname: Lin
authors.affiliation: Department of Optics and Photonics, National Central University, Chung-Li, Taiwan, ROC.
authors.forename: Szu-Yu
authors.initials: SY
authors.lastname: Chen
mesh_terms: D000818:Animals; D017209:Apoptosis; D017077:Culture Media, Conditioned; D004596:Electroretinography; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000502:physiology; D057026:Induced Pluripotent Stem Cells / Q000637:transplantation*; D000090062:Kruppel-Like Factor 4; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000473:pathology / Q000528:radiation effects*
chemical_list: D017077:Culture Media, Conditioned; D051100:Glial Cell Line-Derived Neurotrophic Factor; C000715468:Klf4 protein, mouse; C000715469:Klf4 protein, rat; D000090062:Kruppel-Like Factor 4
keywords: conditional medium; induced pluripotent stem cell; light damage of retina