title: Adult mice transplanted with embryonic retinal progenitor cells: new approach for repairing damaged optic nerves.
abstract: OBJECTIVE
  Retinal ganglion cell (RGC) death and optic nerve degeneration are complex processes whose underlying molecular mechanisms are only vaguely understood. Treatments commonly used for optic nerve degeneration have little long-term value and only prolong degeneration. Recent advances in stem cell replacement therapy offer new ways to overcome RGC loss by transferring healthy cells into eyes of afflicted individuals. However, studies on stem cell replacement for optic nerve degeneration are hampered by limitations of the available animal models, especially genetic models. We have developed a mouse model in which RGCs are genetically ablated in adult mice with subsequent degeneration of the optic nerve. In the study reported here, we used this model to determine whether embryonic retinal progenitor cells (RPCs) removed from donor retinas when RPCs are committing to an RGC fate could restore lost RGCs.
  
  
  METHODS
  We used the RGC-depleted model as a host for transplanting donor green fluorescent protein (GFP)-labeled RPCs from embryonic retinas that are maximally expressing Atoh7, a basic helix-loop-helix gene essential for RGC specification. Dissociated GFP-labeled RPCs were characterized in situ by immunolabeling with antibodies against proteins known to be expressed in RPCs at embryonic day (E)14.5. Dissociated retinal cells were injected into the vitreous of one eye of RGC-depleted mice at two to six months of age. The injected and non-injected retinas were analyzed for gene expression using immunolabeling, and the morphology of optic nerves was assessed visually and with histological staining at different times up to four months after injection.
  
  
  RESULTS
  We demonstrate the successful transfer of embryonic GFP-labeled RPCs into the eyes of RGC-depleted mice. Many transplanted RPCs invaded the ganglion cell layer, but the efficiency of the invasion was low. GFP-labeled cells within the ganglion cell layer expressed genes associated with early and late stages of RGC differentiation, including Pou4f1, Pou4f2, NFL, Map2, and syntaxin. Several GFP-labeled cells were detected within the injected optic nerves of RGC-depleted mice, and in most cases, we observed a significant increase in the thickness of the RPC-injected optic nerves compared with non-injected controls. We also observed more bundled axons emanating from RPC-injected retinas compared with RGC-depleted controls.
  
  
  CONCLUSIONS
  The results offer a new approach for regenerating damaged optic nerves and indicate that a significant number of E14.5 RPCs are able to differentiate into RGCs in the foreign environment of the adult retina. However, the proportion of RPCs that populated the ganglion cell layer and contributed to the optic nerve was not sufficient to account for the increased thickness and higher number of axons. The results support the hypothesis that the injected E14.5 RPCs are contributing autonomously and non-autonomously to restoring damaged optic nerves.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: Jang-Hyeon
authors.initials: JH
authors.lastname: Cho
authors.forename: Chai-An
authors.initials: CA
authors.lastname: Mao
authors.forename: William H
authors.initials: WH
authors.lastname: Klein
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D004622:Embryo, Mammalian; D053595:Embryonic Stem Cells / Q000637:transplantation*; D005260:Female; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D058449:Intravitreal Injections; D051379:Mice; D008822:Mice, Transgenic; D009410:Nerve Degeneration / Q000473:pathology / Q000628:therapy*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009900:Optic Nerve / Q000473:pathology*; D012165:Retinal Ganglion Cells / Q000473:pathology*
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D015415:Biomarkers; D049452:Green Fluorescent Proteins; D009419:Nerve Tissue Proteins; C117638:Atoh7 protein, mouse