EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: Transplantation of stem cells is one of the approaches to treat retinal diseases. Our objective was to determine whether adipose-derived stem cell transplant can survive and migrate in the injured retina using a sodium iodate model for the pigmented retinal epithelium injury.

Materials and Methods: The adipose-derived stem cells were isolated from male albino Sprague-Dawley rats and labeled with DiI so as to track the transplants in the subretinal space. Retinal pigmented epithelium damage was induced by retro-orbital sinus sodium iodate injection (40 mg/kg) into albino Sprague-Dawley rats. Four weeks after transplantation, the eyeballs were fixed in 4% paraformaldehyde and cut with cryostat. The eyeballs were serially sectioned along the vertical meridian. Cryosections were from the full length of the retina and passing through the optic nerve head. The survival and migration of transplanted cells were assessed.

Results: Sodium iodate selectively destroyed the retinal pigmented epithelium layer. The transplanted cells incorporated into the retinal pigmented epithelium layer, perhaps differentiating into a retinal pigmented epithelium phenotype. The transplanted cells were located in the subretinal space; after 4 weeks, some were observed in the retinal pigmented epithelium layer.

Conclusions: We found that adipose-derived stem cells survived for 4 weeks after transplantation and migrated into the retinal pigmented epithelium layer.

Key words : Cytotherapy, Eye disorders, Retinal disease, Retinal pigmented epithelium layer

Retinal pigment epithelium (RPE) dysfunction has been linked to many devastating eye disorders, including age-related macular degeneration and hereditary disorders such as Stargardt disease and retinitis pigmentosa.¹ The integrity of the RPE is essential for retinal function and visual health. The RPE consists of a monolayer of cuboidal cells that separates the photoreceptors and the choroid, forming the blood-retina barrier via tight junctions. It serves an integral function in the visual cycle by phagocytosis of the rod and the cone outer segments following shedding into the subretinal space and the production of paracrine factors for the retina. The RPE is also responsible for movement of ions and water so as to maintain a proper state of dehydration for visual clarity, and its pigmentation absorbs stray light that would otherwise degrade the visual image.^2,3 Pigmentation of the RPE layer is due to the presence of melanosomes, which are organelles containing the light-absorbing pigment melanin. The retinal pigment cells are considered to be postmitotic cells at normal conditions.⁴ They can regenerate and proliferate in an injured retina.⁵ In addition, a low dose of sodium iodate could induce a regenerative mechanism⁶; however, failure of regeneration has been reported with high doses, which could be due to either the retinal stem cells⁷ or the marginal RPE cells.⁸ Therefore, a high-dose model can be useful in evaluating replacement cell therapy for restoring lost cells. However, the success of stem cell therapy is highly dependent on the ability of donor cells to survive, migrate, differentiate, and integrate into the desired location.

On the other hand, mesenchymal stem cells, multipotent cells, are a potential autologous source without immunologic rejection or tumor formation risk; these cells can differentiate into mesodermal, endodermal, and ectodermal lineages such as neurons.^9,10 They can be obtained from bone marrow, but the technique is painful. An alternative source is use of adipose-derived stem cells (ADSCs). These cells are easy to reach, can produce high yield, and can differentiate into different types of cells.¹¹

The purpose of the study was to use ADSCs and to evaluate their survival and migration in the injured RPE developed by injection of sodium iodate.

Materials and Methods

Experimental groups
Twenty-four Sprague-Dawley albino rats (Razi Institute, Tehran, Iran) weighing 250 to 300 g were used in the experiment. The rats were housed in a standard laboratory environment with a 12:12-hour light-dark cycle at 21°C. All experimental procedures involving the animals were performed according to the Association for Research in Vision and Ophthalmology regulations for use of animals in ophthalmic and vision research. The experiments were approved by the Tarbiat Modares University ethics committee.

The rats were divided into 3 groups: the first received subretinal injection of ADSCs (n = 7), and the second group served as the untreated control group (n = 7). All rats received injection of sodium iodate into the retro-orbital sinus. The animals were maintained for 30 days. However, a third group of rats was kept for 1 week after sodium iodate injection (n = 7). The animals in the first group were prepared for cell transplantation.

Damage of the retinal pigment epithelium with sodium iodate
Sodium iodate (Sigma, St. Louis, MO, USA) was diluted in sterile phosphate-buffered saline (PBS) to 2.5 μg/mL and stored at 4°C as stock. The animals were anesthetized by intraperitoneal injection of ketamine (40 mg/kg) and xylazine (4 mg/kg). Pupillary dilation was achieved with 0.5% tropicamide and 0.5% phenylephrine eye drop (Santen, Osaka, Japan). After complete dilatation of the pupil, NaIO3 was injected using an insulin needle into the retro-orbital venous plexus. In brief, we retracted the skin toward the body, causing the eye to protrude, and inserted the needle bevel up into the medial canthus of the eye at a 45-degree angle to the nose into the vessels behind the eye ball. We then gently injected the agent into the retro-orbital vessels and applied light pressure to the eye so as to control bleeding. Thirty days after injection, RPE degeneration was evaluated with whole-mount (RPE layer with remnant of choroid layer) and cross-section methods. Briefly, the eyes were enucleated, and, after brief fixation, the anterior segment was removed and the posterior segment was relaxed onto a slide with 4 radial incisions from the periphery to the optic nerve. The retina was removed, and the RPE layer was peeled from the choroid layer and placed onto a glass slide. The RPE was mounted and observed under a fluorescent microscope. The patchy, loose portions of RPE cells and the accumulation of lipofuscin pigments in the cytoplasm of the RPE cells were investigated and compared with the untreated controls. For tissue cross sections, after enucleation of the eyes, samples were embedded with optimal cutting temperature compound (Sakura Finetek, Zoeterwoude, The Netherlands), which were sectioned (20 μm thickness) with cryostat and stained with hematoxylin and eosin. To investigate outer nuclear layer cell loss, we counted the nuclei of the cells in this layer by ImageJ software (1.45m; National Institutes of Health, Bethesda, MD, USA) in the treated and the untreated control groups in 100-µm sections obtained from the optic disc. We used SPSS software for analyses (SPSS: An IBM Company, version 16.0, IBM Corporation, Armonk, NY, USA).

Cell culture and labeling
The perinephric fat was removed and placed in sterile bottles. Blood was removed, and the fat was minced, transferred to a petri dish, digested in a 50-mL tube containing 20 mL of DMEM culture medium with 0.075% collagenase, and incubated in a shaking incubator (IKA KS 4000i control, Staufen, Germany) at 37°C for 30 minutes. The digests were centrifuged at 400g for 10 minutes, and the supernatant and the fat layers were discarded, whereas the pellet was filtered with a 100-μm mesh cell strainer. The filtrate was recentrifuged at 400g for 10 minutes, and the supernatant was discarded. Each pellet was resuspended in 5 mL of growth medium and filtered through a 70-μm mesh cell strainer. For sample preparations, an additional 20 mL of the growth medium was added to the tube, before the cells were seeded in T75 flasks (25 mL of cell solution/flask). At this point, the yield and the viability of the nucleated cells were determined by staining with trypan blue and counting in hemocytometer. The cells were transferred to a CO2 incubator overnight. After 24 hours, the medium was changed and the nonadherent cells were removed.⁹ For DiI labeling and tracking, the ADSCs of passage 3 (80% confluency) were incubated in cell-labeling solution (6 μL/mL of cell suspension) at 37°C for 30 minutes¹² and then rinsed with PBS. After centrifugation (1500g, 3 min), the supernatant was discarded and the cell pellet was resuspended in the medium for transplantation.

Transplantation of adipose-derived stem cells into the subretinal space
The rats were injected with 40 mg/kg of sodium iodate (Sigma) in the retro-orbital sinus, and approximately 30 days later the animals were prepared for cell transplantation. Rats were anesthetized with intraperitoneal 2% pentobarbital (40 mg/kg; Santen, Osaka, Japan) and topical 1% proparacaine eye drops (Santen). Pupillary dilation was achieved with 0.5% tropicamide and 0.5% phenylephrine eye drops (Santen). After complete dilation, the anesthetized animal was placed in lateral recumbency under Zeiss dissecting microscope and positioned with one holding hand. The rat fundus was visualized with the application of a drop of 2.5% methylcellulose to the eye. The cornea was carefully punctured nasally approximately 0.5 to 1 mm medial to the dilated pupillary margin with a 28-gauge hypodermic needle (Becton Dickinson & Company, Franklin Lakes, NJ, USA). The needle with the bevel up was advanced through the full thickness of cornea into the anterior chamber parallel to the anterior lens face. At least 50% of the bevel was pushed through the cornea, producing a hole sufficiently large enough to insert the 33-gauge blunt needle (Hamilton Company, Reno, NV, USA). The blunt needle tip was inserted through the corneal puncture and advanced into the anterior chamber, avoiding trauma to the iris and lens. Subsequently, the needle shaft was aimed slightly nasally toward the posterior chamber with the iris lateral and lens medial. The lens was displaced medially as the needle was advanced toward the desired injection location. A slight resistance to the movement of the needle indicated penetration of the retina and entrance into the subretinal matrix. At this time, the syringe was held in place, while an assistant pushed the 10-μL ADSC suspension (107 cells/mL) slowly over approximately 30 seconds, injecting the contents of the syringe into the subretinal matrix and creating a visible retinal detachment. After subretinal delivery, the needle was gently withdrawn.^13-15

Immunocytochemistry
The following markers have been evaluated, including ADSCs: mouse anti-CD29 monoclonal antibody (1:400; Abcam, Cambridge, UK) and mouse anti-CD105 monoclonal antibody (1:400; Abcam), which was followed by incubation with antimouse secondary antibody conjugated with fluorescein isothiocyanate.

Tracking the cells using fluorochrome
The cells were labeled with DiI (1,1-dioctadecyl-3,3,3,3’-,cytosine β-D arabinofuranoside; Sigma) before transplantation and counterstained with 4',6-diamidino-2-phenylindole to allow detection after transplantation.

Tissue preparation and immunohistochemistry
The eyeballs were fixed in 4% paraformaldehyde for 2 hours, infiltrated with 30% sucrose overnight at 4°C, and then embedded in optimal cutting temperature compound. Eyeballs were serially sectioned along the vertical meridian. Cryosections, including a full length of retina passing through the optic nerve head and the superior and inferior regions of the eye, were chosen for comparison among the groups. For immunohistochemistry, the sections near the site of injection that had DiI-labeled cells were left to warm at room temperature for 5 minutes, fixed with precooled fixative for 5 to 10 minutes at room temperature, rinsed 3 to 4 times in PBS, and then observed with fluorescent microscope. For pathologic investigation, sections from the transplantation site were air-dried for several minutes so as to remove moisture, stained with 0.1% Mayer hematoxylin (Sigma; MHS-16) for 10 minutes, rinsed in cool running ddH2O for 5 minutes, dipped in 0.5% eosin 12 times, dehydrated, and mounted.

Adipose-derived stem cell culture and DiI labeling
Mesodermal-lineage ADSCs were confirmed by adipogenic and osteogenic differentiation (Figure 1, A and B). The phase-contrast image (Figure 1C) shows ADSCs having typical spindle shape. The ADSC cell markers were evaluated using immuno-cytochemical techniques, with passage 3 cells seeded in 6-well plates. Samples were immunostained with anti-CD29 and anti-CD105 (Figure 1, D and E). Adipose-derived stem cells were labeled with DiI fluorochrome dye for transplant (Figure 1F).

Retinal pigmented epithelium damage model
Administration of sodium iodate, known to selectively damage the RPE, results in patchy loss of RPE and subsequent degeneration of photoreceptors. In our investigation, we used sodium iodate at the retro-orbital venous plexus, with animals maintained for 30 days after injection. After 1 week, the RPE layer showed degenerative changes, with retinal pigment cells showing detachment at low resolution with 4',6-diamidino-2-phenylindole counterstaining (Figure 2). Figure 3 shows changes in the retina after 30 days using whole-mount technique where the accumulation of the lipofuscin is obvious (Figure 3A). An increase in the intercellular space is shown in Figure 3B. The cross sections from the retina (stained with hematoxylin and eosin) are shown at low and high magnification in Figure 3D and Figure 3E. The inner nuclear layer and outer nuclear layer showed disruption in nuclei arrangement, with reduced thickness of the inner nuclear layer. The higher magnification view (Figure 3E) shows degenerative changes in the photoreceptors. There is a significant decrease in the number of nuclei per field in the outer nuclear layer in sodium iodate-treated animals (28 days) compared with that shown in the control group (P < .05).

Survival of transplanted adipose-derived stem cells in the subretinal space
To determine the fate of ADSC transplantation in RPE damage with sodium iodate, cells were labeled with DiI injected into the subretinal space. The distribution of the labeled ADSCs was achieved by evaluating retinal tissue 4 weeks after trans-plantation, with results showing some cells located in the subretinal space (Figure 4A, D, H, J, and M; Figure 5A-F; Figure 6A-F; Figure 7B-C, G-H). As shown in Figure 6D-H, intact nuclei indicate viable cells where ADSCs survived after 1 month of transplantation. Some cells (Figure 7G and 7H) were located in the subretinal space with large nuclei, whereas some areas showed binucleated-like cells with 2 small nucleus-like cells, which may be related to dividing cells at the telophase.

Migration of the transplanted adipose-derived stem cells into the retinal tissue
As shown in Figure 4M and 4N, labeled cells (on retinal whole mount) crossed the retinal tissues from subretinal space and located in the RPE layer or deeper into the retina, shown in the merged images (Figure 4M-O). Migration of labeled transplants into the sensory retina is also visible in Figure 5A-C, versus those that located in the subretinal space (Figure 5D-F). In addition, as shown in Figure 6, labeled transplants crossed into the retinal tissue and located at the RPE layer (Figure 5C and Figure 6A-F), with some labeled cells penetrating deeper in the sensory retina (Figure 5C and Figure 6F). Hematoxylin and eosin-stained sections showed cells that crossed the retinal tissue from the subretinal space (Figure 4P). As shown in Figure 6H and 6I, transplanted cells also transgressed the retinal tissue toward the sensory retina from the same section used for detection of the labeled areas on fluorescence microscope (stained with hematoxylin and eosin stains). Hematoxylin and eosin-stained sections of transplanted cells were also located in the subretinal space (Figure 7A-D and Figure 7G and 7H), with migration in the retinal tissue at the choroid layer (Figure 7E and 7F) and deeper in the retinal tissue (Figure 7I-L).

In this study, we used sodium iodate to induce damaged RPE by retro-orbital injection. Other investigators have used other routes of drug delivery, including tail vein, intraperitoneally, and through the lingual vein,^6,16,17 with oxidative stress also used as a mechanism of RPE damage.¹⁸ Subretinal delivery of ADSCs resulted in their survival and migration in the retina, suggesting that cell therapy can be considered as a possible option for treating damaged RPE. Cell replacement of the degenerated RPE in dry-type age-related macular degeneration was suggested by other investigators.^19,20

The delivered stem cells should tolerate the new milieu, survive, migrate to the lesion site, dif-ferentiate, and functionally integrate in the retina, resulting in restoration of the lost RPE layer.²¹ The possible mechanisms of ADSC migration in damaged retina include expression of chemoattractants²² such as SDF-1, which could interact with its receptor CXCR4 in the ADSCs, resulting in their migration.²³ On the other hand, ADSCs, multipotent adult stem cells,⁹ have been used in this investigation as a source for cytotherapy. Other sources such as embryonic stem cells were also successfully used, with animal models showing signs of improvement.²¹ The ethical issue of using embryonic stem cells remains a challenge as the source of these cells is the blastocyst of an embryo. This allogenic source can raise concern about immunologic rejection; moreover, tumo-rigenesis was reported in transplantations with embryonic stem cells.²⁴ Induced pluripotent stem cells are also a feasible source for cell therapy; however, their tumorigenesis was documented,^20,25 such as that shown with malignant germ cell-like tumors.²⁶ Therefore, adult stem cells such as ADSCs are safe sources for transplantation.²⁷ In this study, the survival and migration of the ADSCs were visible in damaged RPE. The survival and integration of ADSCs were reported in normal retina^28,29 and had been injected through the sclera to deliver them to the vitreous cavity.³⁰ Moreover, transplanted ADSCs into a developing mouse eye were reported by other investigators,³¹ which integrated and differentiated in the host eye, forming cells immunoreactive to microtubule-associated protein 2 (a marker for mature neuron in the ganglion cell layer) or glial fibrillary acid protein (a marker of astrocytes), suggesting that ADSCs can adapt to the retinal microenvironment.³¹ Other investigators empha-sized the protective effects of these cells; others reported that ADSCs could protect the structure and function of the retina from the light-induced damage by progranulin, a secretory protein from ADSCs, in in vitro and in vivo models.³² Others have documented that they could rescue the retinal neurons from the degenerative effects of hyperglycemia in a diabetic model, resulting in improved visual function.³³ Therefore, the migration of ADSCs can improve the visual function in a degenerated retina by replacing the lost RPE or by protecting the degenerating ones or both.

Another source for autotransplant is bone marrow stromal cells, which have been used in rodent models of retinal degeneration. Bone marrow-derived stem cells may restore the function of the retina through different mechanisms: cellular differentiation, paracrine effect, and retinal pigment epithelium repair.^17,34-36 However, there are few studies on the use of ADSCs in transplantation. Bone marrow stromal cell subretinal transplantation could inhibit photoreceptor apoptosis and slow down retinal damage in light-damaged eyes, where they could express basic fibroblast growth factor (in vitro) and brain-derived neurotrophic factor (in vitro and in vivo), pointing to the potential trophic and protective effects on light-damaged retinas.³⁷ Yang and associates showed that both endogenous and exogenous bone marrow stromal cells could incorporate to the RPE layer and express RPE linage markers such as RPE65 and MITF.⁶ In this study, DiI fluorochrome was used, as DiI was shown to be nontoxic, highly stable, and efficient for steady tracing of cells compared with bromodeoxyuridine.³⁸

Volume: 16 Issue: 2 April 2018

FULL TEXT