Diabetic retinopathy is the most common diabetic eye disease and a leading cause of blindness in American adults.1 It is well established that in diabetic patients, sustained high glucose levels cause blood vessel damage, leading to increased vascular permeability, macular edema, and ischemia in the retina.2 This further induces neovascularization and bleeding in the eye, resulting in retinal neuron loss and eventual blindness. Laser treatment, intravitreal injections, and vitrectomy are effective treatments, but many patients nonetheless progress to severe visual loss.
Recently, adult retinal stem cells (RSCs) have been discovered in adult mammalian eyes, reinvigorating the regenerative strategy for the treatment of retinal degeneration.3,4 We have found that pigment epithelium-derived factor (PEDF), a potent inhibitor of angiogenesis and vascular permeability, is expressed in RSC spheres. These developments have led us to hypothesize that transplantation of RSCs may prevent or slow down the vascular damage and neovascularization in diabetic retinopathy, as well as provide new retinal cells to replenish those retinal neurons lost to disease.5,6
METHODS AND INVESTIGATION
In our studies, we used Ins-2Akita/wt (Akita) mice with a dominant mutation in one allele of the insulin-2 gene as a diabetic retinopathy mouse model. We isolated the RSCs from adult Tg(ACTB enhanced yellow fluorescent protein [EYFP]) mice,7 in which all cells express EYFP. The RSCs were isolated from the ciliary epithelium and cultured in chemically defined serum free media for at least 7 days. Approximately 1 in 500 of the ciliary epithelium cells grows into the RSC spheres.3 We then dissociated the RSC spheres into single-cell suspension in Hank's Balanced Salt Solution (HBSS) for transplantation.
The disassociated cells were injected into the vitreous of the right eye of the Akita mouse—about 20,000 cells/0.5 µL per injection—and the left eye was injected with HBSS that did not contain cells for a negative control. Transplanted eyes were collected 1 week, and 1, 3, and 6 months after transplantation. We then studied the survival, integration, and differentiation of the transplanted RSC sphere cells, as well as the morphology and vascular permeability of the host retinas.
FINDINGS
We found that most of the RSC sphere cells were present and alive in the vitreous cavity. The transplanted cells were alive up to 6 months after injection, the longest time-point studied to date. Most of the surviving cells, however continued to stay in the vitreous. These surviving RSC sphere cells appear to retain their progenitor cell identity, but, importantly, they did not overproliferate to form tumors. Further characterization of the integration and differentiation of the transplanted cells, and study of the progression of diabetic retinopathy in the host retina, are currently ongoing.
Part of our continuing work is to study whether these cells are still expressing PEDF and whether RSC sphere cells can migrate into the retina and differentiate into functional mature retinal cells. It is possible that these transplanted cells are beneficial to alleviate diabetic retinopathy by existing in the vitreous, because they may still produce PEDF and other factiors to prevent vascular leakage and neovascularization.
BUILDING ON ADDITIONAL RESEARCH
Recently, we successfully achieved robust integration and differentiation of the retinal progenitor cells in the adult mouse retina, similar to the findings of the integration and differentiation of postmitotic precursor cells into functional rod photoreceptors in the host retina after subretinal transplantation reported by MacLaren et al.8,9 Currently, we are working on further improvement in the migration, integration, and differentiation of the RSC spheres in the host retina, and on the detailed analysis of secreted factors that RSC sphere cells may produce after transplantation.
With improved integration and differentiation, transplanted RSC sphere cells can potentially provide dual beneficial effects in diabetic retinopathy treatment: They may inhibit abnormal vasopermeability and also supplement new neurons to the degenerated retina. RSC sphere-cell transplantation may be a novel treatment of diabetic retinopathy and other retinal degenerative diseases, including age-related macular degeneration and retinitis pigmentosa.
Beatrix Kovacs, PhD, is a postdoctoral fellow in the Department of Ophthalmology and Neurological Science at Rush University, Chicago.
Mathew W. MacCumber, MD, PhD, is Associate Professor, Associate Chairman for Research, Rush University Medical Center. He may be reached at macretina@aol.com; phone: 773-871-8444; or fax: 773-871-4781.
Shunbin Xu, MD, PhD, is Assistant Professor and directs the laboratory in the Department of Ophthalmology at Rush University. He may be reached at shunbin_xu@rush.edu; phone: 312-563-3554; or fax: 312-563-3571.
This work was supported by a grant from the Juvenile Diabetes Research Foundation, International.
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