Abstract
Purpose: :
Hematopoietic stem cells (HSC) have been shown to have marked plasticity and be involved in repair. Infecting embryonic stem cells with viral vectors expressing terminal differentiation markers can force the cells along a particular differentiation pathway. We asked whether HSC can home to sites of retinal pigment epithelium (RPE) cell injury and become RPE when transduced to express an RPE–specific protein.
Methods: :
The RPE of C57Bl6 mice was injured by systemic injection of sodium iodate. HSC from gfp+/+ transgenic mice were infected ex vivo with a lentiviral vector expressing either LacZ (control) or RPE65. The infected gfp+ HSC were then injected systemically into the sodium iodate–treated C57Bl6 mice, termed adoptive transfer. Another cohort of animals received nontransduced gfp+ HSC. A final cohort received a comparable volume of saline. Immunohistochemistry and histology were used to determine the degree of RPE damage and repair, as well as the presence and location of gfp+ cells, endothelial cells, RPE65 protein, and RPE–specific protein CRALBP. Electroretinograms were also performed to measure retinal function after giving sodium iodate and HSC.
Results: :
As expected, sodium iodate injection resulted in major damage to the RPE and photoreceptors. Gfp+ cells were not seen in mice not given HSC. Uninfected cells and LacZ–infected cells had negligible RPE restoration. However, in the cohort of animals that were given HSC expressing RPE65, there was robust restoration of the RPE layer. In many damaged areas that showed recovery, gfp+CRALBP+ expression was abundant. These gfp+ cells displayed typical RPE morphology.
Conclusions: :
Cells that co–expressed gfp and CRALBP were present in areas of RPE ablation. This confirms that HSC are capable of differentiating into RPE cells following injury. The overexpression of RPE65 is apparently sufficient to drive the differentiation of HSC to RPE. These findings suggest that a novel therapeutic option: that HSC can be modified ex vivo to induce specific differentiation, and then returned to a patient to replace lost or damaged RPE.
Keywords: regeneration • retinal pigment epithelium • gene transfer/gene therapy