Non-invasive stem cell therapy in a rat model for retinal degeneration and vascular pathology

Abstract

Background: Retinitis pigmentosa (RP) is characterized by progressive night blindness, visual field loss, altered vascular permeability and loss of central vision. Currently there is no effective treatment available except gene replacement therapy has shown promise in a few patients with specific gene defects. There is an urgent need to develop therapies that offer generic neuro-and vascular-protective effects with non-invasive intervention. Here we explored the potential of systemic administration of pluripotent bone marrow-derived mesenchymal stem cells (MSCs) to rescue vision and associated vascular pathology in the Royal College Surgeons (RCS) rat, a well-established animal model for RP.

Methodology/principal findings: Animals received syngeneic MSCs (1x10(6) cells) by tail vein at an age before major photoreceptor loss.

Principal results: both rod and cone photoreceptors were preserved (5-6 cells thick) at the time when control animal has a single layer of photoreceptors remained; Visual function was significantly preserved compared with controls as determined by visual acuity and luminance threshold recording from the superior colliculus; The number of pathological vascular complexes (abnormal vessels associated with migrating pigment epithelium cells) and area of vascular leakage that would ordinarily develop were dramatically reduced; Semi-quantitative RT-PCR analysis indicated there was upregulation of growth factors and immunohistochemistry revealed that there was an increase in neurotrophic factors within eyes of animals that received MSCs.

Conclusions/significance: These results underscore the potential application of MSCs in treating retinal degeneration. The advantages of this non-invasive cell-based therapy are: cells are easily isolated and can be expanded in large quantity for autologous graft; hypoimmunogenic nature as allogeneic donors; less controversial in nature than other stem cells; can be readministered with minor discomfort. Therefore, MSCs may prove to be the ideal cell source for auto-cell therapy for retinal degeneration and other ocular vascular diseases.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Rod and cone protection.

A. Retinal sections stained with cresyl violet indicate substantial preservation of photoreceptors across the retina in MSC treated eyes at P90, while in control eyes (sham injected (B) and untreated (C)): only a single layer of photoreceptors remained. A1, A2 &A3: higher power images showing preservation of photoreceptors from the insets A1, A2 &A3 in A. D, E&F: confocal images showing rhodopsin (green in D) and cone arrestin (red in E) positive staining at P90 in MSC treated retina, while in sham injected retina, cone arrestin staining was dramatically reduced (F). All sections were counterstained with DAPI (blue) (scale bars equal 50 µm).

Figure 2. Preservation of visual function.

A. Visual acuity tested by Optomotor response. Unrestrained animals were placed on a platform, where they tracked the grating with reflexive head movements. The acuity threshold was quantified by increasing the spatial frequency of the grating. RCS rats received MSCs and medium injection via tail vein at P30 and tested at P90. Visual acuity was significantly better in MSC treated eyes compared with controls (P

Figure 3. Vascular protection.

A–F: Retinal whole mount was stained with NADPH-diaphorase: A. typical vascular pathology in the eye at P90 in untreated RCS rat: vascular complexes (abnormal vessels associated with RPE cells) were mainly located around the optic nerve disc (arrows) and spread peripheral with age. B. vascular complexes in the middle to peripheral retina (arrows). C. high power image showing vascular complexes (arrows) from B. D. RCS retina treated with MSCs at P90: the vascular complexes were dramatically reduced around the optic nerve disc. E. two vascular complexes (arrows) in the middle field of the retina. F. high power image from E showing vascular complexes (arrow). G–L. animal was perfused with FITC-dextran, whole mount was prepared: G. typical vascular leakage, mainly around the optic disc in untreated eye at P90. H–K. high power images from G showing vascular leakage (arrows in H) and abnormal vessels (arrows in I–K). L. MSC treated retina, the vascular leakage around the optic nerve disc was greatly reduced. M&N. high power images from L showing much reduced leakage (arrows in M) and small abnormal vessels (arrow in N) (Scale bars equal 250 µm for A, D, G &L; 100 µm for F).

Figure 4. Upregulation of trophic factors.

A. Semi-quantitative RT-PCR for CNTF, bFGF, BDNF and beta actin. Lane 1: RNA isolated from MSC prior to injection; Lane 2–4: RNA isolated from retinas treated with MSC; Lane 5–7: RNA isolated from non-treated control retinas. B. Densitometry analysis of CNTF, BDNF and bFGF in treated versus untreated samples. Beta actin was used to normalize the data for comparison. Level of CNTF and BDNF in the treated retinas were significantly higher than non-treated controls (p

Figure 5. Distribution of MSCs.

A. phase contrast microphotograph of bone marrow derived mesenchymal stem cells at passage 2. B. MSCs were preincubated with PKH26 before intravenous injection. C. PKH26 labeled MSCs in the retina two weeks after intravenous injection (arrows); blood vessels were perfused with FITC-dextran (green). D–F. showing PKH26 labeled MSCs in the retinal section (D, arrows pointing PKH26 labeled MSCs; double arrows indicating background staining in debris zone); sections counterstained with DAPI (E); F. merged image from D&E showing PKH26 labeled MSCs counterstained with DAPI (scale bar equals 100 µm).