July 2010
Volume 51, Issue 7
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Retina  |   July 2010
Effects of Bone Marrow Mesenchymal Stem Cell Transplantation on Light-Damaged Retina
Author Affiliations & Notes
  • Yu Zhang
    From the Peking University Third Hospital, Peking University Eye Center, Beijing, China.
  • Wei Wang
    From the Peking University Third Hospital, Peking University Eye Center, Beijing, China.
  • Corresponding author: Wei Wang, Peking University Third Hospital, Peking University Eye Center, 49 Huayuan North Road, Haidian District, Beijing, China 100191; puh3_ww@bjmu.edu.cn
Investigative Ophthalmology & Visual Science July 2010, Vol.51, 3742-3748. doi:10.1167/iovs.08-3314
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      Yu Zhang, Wei Wang; Effects of Bone Marrow Mesenchymal Stem Cell Transplantation on Light-Damaged Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(7):3742-3748. doi: 10.1167/iovs.08-3314.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To investigate the effects and possible mechanisms of rat bone marrow mesenchymal stem cell (BMSC) transplantation on the light-damaged retinal structure and the apoptosis of photoreceptors.

Methods.: DAPI-labeled BMSCs were transplanted into the subretinal space of light-damaged Sprague–Dawley rats 10 days after exposure. BMSCs were cultivated with the supernatant of homogenized retina (SHR).

Results.: The outer nuclear layer (ONL) contained significantly more cells and the percentage of apoptotic ONL cells was significantly reduced in the BMSC transplantation group than in the phosphate-buffered solution injection group or the light damage group. Most DAPI-labeled BMSCs expressed brain-derived neurotrophic factor (BDNF). There was elevated basic fibroblast growth factor (bFGF) and BDNF immunoreactivity in the retinas of the BMSC transplantation group compared with the light damage group. In vitro culture showed that 10% of BMSCs changed from fusiform shape to multipolar shape. A fraction of cells expressed MAP2 or glial fibrillary acidic protein, and some cells expressed bFGF or BDNF when cultivated with light-damaged SHR for 7 days.

Conclusions.: BMSC subretinal transplantation could inhibit photoreceptor apoptosis and slow down retinal damage in light-damaged eyes. BMSCs could express bFGF (in vitro) and BDNF (in vitro and in vivo), pointing to potential trophic and protective effects on light-damaged retinas.

There are no successful therapies for the treatment of photoreceptor degeneration and apoptosis, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Photoreceptor replacement, in the form of a cell-based therapeutic approach, might possibly aid in the restoration of some degree of vision. Embryonic stem cell therapy, though promising, remains highly controversial and must overcome the potential problem of rejection. Thus, because of their autologous characteristics, multipotency, 1,2 relative ease of isolation, and less controversial nature, bone marrow mesenchymal stem cells (BMSCs) have become a forerunner as the cells of choice in cell-based therapy. 
BMSCs can differentiate into neurons or glia, 39 and they can express rhodopsin and opsin, which shows their potential ability to differentiate into photoreceptors. 1013 Moreover, BMSCs are known to express a variety of cytokines and neurotrophic factors, 1416 including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and basic fibroblast growth factor (bFGF), with their powerful trophic and protective functions. BMSC transplantation has been reported to be beneficial in treating several kinds of models of CNS disorders. 1719 BMSC intraocular transplantation can inhibit retinal ganglion cells (RGCs) apoptosis in optic nerve injury or glaucoma models 20,21 and can delay the aggravation of photoreceptor apoptosis in Royal College of Surgeons (RCS) rats. 22,23  
In this study, we used green light to produce retinal light damage to Sprague–Dawley (SD) rats, which primarily caused photoreceptor apoptosis similar to RP and AMD. We investigated whether BMSC transplantation in light-damaged model eyes could prevent the reduction of photoreceptors in the host retina. We also tried to find possible mechanisms in vivo and in vitro. 
Materials and Methods
Reagents
Dulbecco's modified Eagle medium-low glucose (DMEM-LG), fetal bovine serum (FBS), and trypsin-EDTA were purchased from Invitrogen (Melbourne, Australia). DAPI was obtained from Sigma (St. Louis, MO). PharMingen (San Diego, CA) supplied all secondary antibodies and block sera. Fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) kit was obtained from Promega (Madison, WI). 
Animals
Thirty-eight adult female SD rats (weight range, 160–180 g) were obtained from the experimental animal center of Peking University's health department. All animal experiments in this study adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
BMSC Culture, Characterization, and Labeling
Six rats were killed with an overdose of pentobarbital, and their tibias and femurs were dissected. The marrow was then extruded with DMEM-LG. The nucleated cells were counted; suspended at a concentration of 1 × 105/mL in DMEM-LG; supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and plated at 3 × 104/cm2 in 100-mm culture dishes at 37°C, humidity 95%, and CO2 5%. After 24 hours, nonadherent cells were removed by replacement of the medium. Medium was changed every 3 days. Once adherent cells were more than 80% confluent, they were recovered with trypsin-EDTA and replated at a ratio of 1:3. 
The isolated and expanded cells were characterized at passage 2 by immunocytochemistry. Cells were fixed with 4% paraformaldehyde for 15 minutes, washed, and then blocked in goat or rabbit serum for 1 hour at 37°C. Cells were incubated with various primary antibodies (Table 1) for 24 hours at 4°C (negative and positive controls were made simultaneously), followed by secondary antibodies (fluorescein isothiocyanate [FITC] or tetraethyl rhodamine isothiocyanate [TRITC] conjugated) for 1 hour at 37°C. Finally, nuclei were counterstained with Hoechst (1:1000) for 10 minutes at room temperature. The cells were visualized using a fluorescent microscope (Olympus Optical, Tokyo, Japan), and fluorescent images were merged with imaging software (NIS Elements; Nikon, Tokyo, Japan). The immunoreactivity of every marker was identified by one cell slide. Positive percentages of cells in five nonoverlapping fields of view (×200) of each slide were counted, and the average represented the positive rate of each marker. 
Table 1.
 
Primary Antibodies in Immunocytochemistry to Characterize BMSCs
Table 1.
 
Primary Antibodies in Immunocytochemistry to Characterize BMSCs
Primary Antibody Host Titer Cell Supplier
Anti-CD34 Mouse 1:100 Hematopoietic cell Santa Cruz Biotechnology (Santa Cruz, CA)
Anti-CD90 Mouse 1:500 Undifferentiated cell Chemicon (Temecula, CA)
Anti-CD44 Goat 1:100 Stromal cell Santa Cruz Biotechnology
Anti-CD166 Goat 1:100 Stromal cell Santa Cruz Biotechnology
Anti-nestin Mouse 1:50 Neural stem cell Chemicon
Anti-MAP2 Rabbit 1:400 Neuron Chemicon
Anti-rhodopsin Mouse 1:200 Rod photoreceptor Chemicon
Anti-calretinin Mouse 1:1500 Amacrine cell Chemicon
Anti-GFAP Rabbit 1:400 Neuroglial cell Chemicon
Anti-CD11b Mouse 1:50 Phagocyte Chemicon
Anti-bFGF Rabbit 1:1000 Chemicon
Anti-BDNF Rabbit 1:1000 Chemicon
Anti-CNTF Mouse 1:50 Chemicon
DAPI was added into medium at passage 2 with a final concentration of 50 mg/L and was incubated at 37°C for 1 hour. Cells were washed with Hanks balanced salt solution six times, digested with trypsin-EDTA, and suspended in PBS (106 cells /mL). Then 5 μL DAPI-BMSC suspension (5 × 103 cells) was transplanted into each eye. 
Light Damage Model
Adult female SD rats (weight range, 160–180 g) were maintained on a 12-hour light/12-hour dark cycle for 7 days in cages of 60 to 100 lux and had access to food and water ad libitum. After undergoing dark adaptation for 24 hours, rats were placed in a green light environment (range, 480–520 nm) with an in-cage illuminance level of 950 ± 50 lux for 24 hours. Control rats were maintained in weak white light conditions of 60 to 100 lux. After such exposure, rats underwent dark adaptation for 3 days. 
Animal Grouping
Twenty-four SD rats were randomly divided into a normal control group, a light damage group, a PBS injection group, and a BMSC transplantation group with six rats in each group. The operations were done on the 10th day after light exposure in the latter two groups. On the 24th day after exposure (14 days after operation), all rats were killed, and the eyes were enucleated. 
Subretinal Transplantation
Rats were anesthetized with intraperitoneal 2% pentobarbital (40 mg/kg) and topical 1% proparacaine eye drops (Santen, Osaka, Japan). Pupillary dilation was achieved with 0.5% tropicamide and 0.5% phenylephrine eye drops (Santen). Guided by an operating microscope, a superior conjunctival incision was made, and a modified 10-μL Hamilton microsyringe was used to pierce the sclera and choroids 2 mm posterior to the superior limbus in the right eye. Subsequently, 5 μL BMSC suspension (106 cells/mL) or 5 μL PBS was slowly injected into the subretinal space that detached nearly half the retina. After IOP was decreased by puncturing the anterior chamber, the syringe was plucked, and the conjunctival incision was electrically coagulated. 
Immunohistochemistry
Eyeballs were fixed in 4% paraformaldehyde for 2 hours, infiltrated with 30% sucrose overnight at 4°C, and then embedded in OCT. Eyeballs were continuously 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 groups. Immunohistochemistry on cryosections that included DAPI-positive cells were performed to detect the differentiation of transplanted BMSCs. Cryosections were blocked with goat serum for 1 hour at 37°C and then incubated with primary antibodies for 24 hours at 4°C, followed by secondary antibodies for 1 hour at 37°C. Primary antibodies included rabbit polyclonal antibody against rat microtubule-associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP), bFGF, and BDNF and mouse monoclonal antibody against nestin, rhodopsin, calretinin, and CNTF. 
TUNEL Detection
Cryosections, including the optic nerve head, were washed and dipped into methanol for 10 minutes at −20°C. After they were covered with 100 μL equilibration buffer for 10 minutes, the tissues were incubated with reaction compound (equilibration buffer/nucleotide mix/terminal deoxynucleotidyl transferase, 45:5:1) for 1 hour at 37°C. Tissues were covered with 2× SSC to end the reaction, washed, and incubated with Hoechst (1:1000) for 10 minutes at room temperature. 
Supernatant of Homogenized Retina Preparation
Four normal SD rats and four light-damaged SD rats (10 days after exposure) were used for the preparation of supernatant of homogenized retina (SHR). Eight normal neural retinas and eight light-damaged neural retinas were separately homogenized in 2 mL DMEM-LG. Homogenates were centrifuged at 12,000 rpm for 20 minutes at 4°C, and the final clear supernatant was obtained. Immediately, the total protein content of SHR was analyzed using background-corrected absorbance (BCA; standard protein was diluted to a gradient concentration from 0 to 2 mg/mL by DMEM-LG, BCA fluid was added to the diluted standard protein and SHR in 96-well orifice for 30 minutes at 37°, A570 was determined by spectrophotometer, and the protein content of SHR was calculated by standard curve). SHR was stored as aliquots at −80°C. 
BMSC Cultivation with SHR
At passage 2, the cells were cultured with DMEM-LG for 24 hours in a 12-well orifice (105 cells/well). Then normal SHR (protein content, 1200 μg/mL) or light-damaged SHR (protein content, 750 μg/mL) was added into DMEM-LG media with a final SHR protein content of 200 μg/mL for 1, 3, or 7 days. The control media were DMEM-LG without SHR. 
Differentiation and Cytokine Expression of BMSCs Cultivated with SHR
Cultivated cells were identified by immunocytochemistry. Primary antibodies included rabbit polyclonal antibody against rat MAP2, GFAP, bFGF, and BDNF and mouse monoclonal antibody against nestin, rhodopsin, and CNTF. Each instance of antigen immunoreactivity was identified by one cell slide. Positive percentages of cells in five nonoverlapping fields of view (×200) of each cell-slide were counted, and the average represented the positive rate of each protein. 
Statistical Analysis
Retinal areas of 2 to 3 disc diameters (DD) distance superior to the optic nerve head (S2–S3) in cross-sections passing though the optic disc were taken for comparison. The number of outer nuclear layer (ONL) cells and the percentage of apoptotic ONL cells on five random slices of each eye were averaged and were considered as 2 indexes of each eye. The two indexes among groups were compared by ANOVA using data analysis software (SPSS 11.0; SPSS, Chicago IL). Statistical significance was declared at P < 0.05. 
Results
Morphology and Characterization of BMSCs
Twenty-four hours after the initial seeding, a few adherent fibroblastic-like mononuclear cells were observed. Three days later, adherent cells increased obviously and took on a colony-like look. Cells reached confluence 7 to 10 days later. At passage 2, adherent cells became uniform and grew in whirlpool, radial, or parallel patterns. 
Immunocytochemistry indicated that all cells were negative for CD34, and most cells were positive for CD44 (98.5%), CD166 (95.6%), and CD90 (94.2%). A small percentage (0.2%) of cells was positive for nestin. A few (1.5%) small nuclear cells were positive for CD11b. All cells were negative for MAP2, rhodopsin, calretinin, and GFAP. Almost all cells expressed bFGF (99.5%), CNTF (98.4%), and BDNF (99.8%; Fig. 1). DAPI showed positive in the nuclei of almost all the cells, which emitted blue fluorescence under ultraviolet light. 
Figure 1.
 
Characterization of P2-BMSCs by immunocytochemistry. Expression of (A) CD44, (B) CD166, (C) CD90, (D) Nestin, (E) CD11b, (F) bFGF, (G) CNTF, and (H) BDNF by BMSCs. CD44, CD166, and CD11b immunoreactivities (green) were detected by FITC-labeled secondary antibody, and the others immunoreactivities (red) were detected by TRITC-labeled secondary antibody. (AE) Cell nuclei (blue) were counterstained with Hoechst. Immunoreactivity of CD44, CD166, and CD90 was 98.5%, 95.6%, and 94.2%, respectively. 0.2% BMSCs expressed nestin and 1.5% BMSCs expressed CD11b. Scale bar, 100 μm.
Figure 1.
 
Characterization of P2-BMSCs by immunocytochemistry. Expression of (A) CD44, (B) CD166, (C) CD90, (D) Nestin, (E) CD11b, (F) bFGF, (G) CNTF, and (H) BDNF by BMSCs. CD44, CD166, and CD11b immunoreactivities (green) were detected by FITC-labeled secondary antibody, and the others immunoreactivities (red) were detected by TRITC-labeled secondary antibody. (AE) Cell nuclei (blue) were counterstained with Hoechst. Immunoreactivity of CD44, CD166, and CD90 was 98.5%, 95.6%, and 94.2%, respectively. 0.2% BMSCs expressed nestin and 1.5% BMSCs expressed CD11b. Scale bar, 100 μm.
Comparison of Retinal Structure among Groups
After injury, the outer and inner segments of most photoreceptors shortened and even vanished gradually. The number of ONL cells decreased as time went on. The most severely affected location was at S2 to S3, where the number of ONL cells showed a significant difference between groups (P < 0.05) 24 days after light damage (normal group, 1020.9 ± 148.2 cells; light damage group, 40.2 ± 40.5 cells; PBS injection group, 365.5 ± 100.8 cells; BMSC transplantation group, 690.0 ± 114.6 cells; Fig. 2). 
Figure 2.
 
Comparison of S2–S3 retina structure among groups (hematoxylin and eosin stain). The number of ONL cells showed a significant difference between groups (P < 0.05, analysis of variance). (A) Normal eye. (B) In the light-damaged eye (24 days after light damage), the ONL cells decreased significantly. (C) The number of ONL cells was greater in the PBS-injected eye (24 days after light damage and 14 days after PBS injection) than in light-damaged eye. (D) The number of ONL cells was greater in the BMSC-transplanted eye (24 days after light damage and 14 days after BMSC transplantation) than in PBS injected eye. Scale bar, 100 μm. S2–S3 retina, retina 2 to 3 DD distance superior to the optic disc.
Figure 2.
 
Comparison of S2–S3 retina structure among groups (hematoxylin and eosin stain). The number of ONL cells showed a significant difference between groups (P < 0.05, analysis of variance). (A) Normal eye. (B) In the light-damaged eye (24 days after light damage), the ONL cells decreased significantly. (C) The number of ONL cells was greater in the PBS-injected eye (24 days after light damage and 14 days after PBS injection) than in light-damaged eye. (D) The number of ONL cells was greater in the BMSC-transplanted eye (24 days after light damage and 14 days after BMSC transplantation) than in PBS injected eye. Scale bar, 100 μm. S2–S3 retina, retina 2 to 3 DD distance superior to the optic disc.
Comparison of ONL Cells Apoptosis among Groups
After light damage, apoptosis happened primarily in the ONL of the superior retina. Retinas of S2 to S3 were taken for comparison to count the mean percentages of apoptotic ONL cells 24 days after light damage (normal group, 0.10% ± 0.05%; light damage group, 9.17% ± 1.23%; PBS injection group, 2.19% ± 0.57%; BMSC transplantation group, 0.16% ± 0.09%). The BMSC transplantation group had a smaller percentage of apoptotic ONL cells than did the PBS injection group or the light damage group (P < 0.05). The PBS injection group also had a smaller percentage of apoptotic cells than did the light damage group (P < 0.05). However, the BMSC transplantation group had no significant difference from the normal group (P > 0.05; Fig. 3). 
Figure 3.
 
Comparison of apoptosis of S2–S3retina among groups. Apoptosis of ONL cells in each group was detected by TUNEL analysis (green) 24 days after light damage. Cell nuclei (blue) were counterstained with Hoechst. (A) Normal eye. (B) Light-damaged eye. (C) The percentage of apoptotic ONL cells was smaller in the PBS-injected eye (14 days after PBS injection) than in the light-damaged eye (P < 0.05). (D) The percentage of apoptotic ONL cells was smaller in the BMSC-transplanted eye (14 days after BMSC transplantation) than in the PBS-injected eye (P < 0.05), but the difference was not significant compared with the normal group (P > 0.05). Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Figure 3.
 
Comparison of apoptosis of S2–S3retina among groups. Apoptosis of ONL cells in each group was detected by TUNEL analysis (green) 24 days after light damage. Cell nuclei (blue) were counterstained with Hoechst. (A) Normal eye. (B) Light-damaged eye. (C) The percentage of apoptotic ONL cells was smaller in the PBS-injected eye (14 days after PBS injection) than in the light-damaged eye (P < 0.05). (D) The percentage of apoptotic ONL cells was smaller in the BMSC-transplanted eye (14 days after BMSC transplantation) than in the PBS-injected eye (P < 0.05), but the difference was not significant compared with the normal group (P > 0.05). Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Cytokine Immunoreactivity of S2 to S3 Retinas
BFGF immunoreactivity was normally located within cells in the inner nuclear layer (INL) but was lowered in light-damaged retinas. bFGF immunoreactivity was elevated in the INL and ONL in the BMSC transplantation group and the PBS injection group; this immunoreactivity was more intense in the former group (Fig. 4). BDNF immunoreactivity was located within cells in the ganglion cell layer (GCL) and the INL in all groups. There was no difference between the normal and light damage group. BDNF immunoreactivity was similarly elevated in the BMSC group and the PBS group compared with the normal and light damage groups (Fig. 5). CNTF immunoreactivity was faintly detected in the normal group but became intense in the INL and GCL in the light damage group. There was lowered CNTF immunoreactivity in the BMSC group and the PBS group compared with the light damage group, primarily in the GCL (Fig. 6). 
Figure 4.
 
Comparison of bFGF immunoreactivity of S2–S3 retinas among groups. Expression of bFGF in retina was identified by rabbit polyclonal anti–rat bFGF antibody. BFGF immunoreactivity (red) was detected by TRITC-labeled anti–rabbit antibody. (A) In the normal eye, bFGF immunoreactivity was located in the INL. (B) In the light-damaged eye, bFGF was lowered. bFGF immunoreactivity was elevated in the INL and ONL in the PBS-injected eye (C) and the BMSC-transplanted eye (D), the latter of which experienced greater immunoreactivity. Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Figure 4.
 
Comparison of bFGF immunoreactivity of S2–S3 retinas among groups. Expression of bFGF in retina was identified by rabbit polyclonal anti–rat bFGF antibody. BFGF immunoreactivity (red) was detected by TRITC-labeled anti–rabbit antibody. (A) In the normal eye, bFGF immunoreactivity was located in the INL. (B) In the light-damaged eye, bFGF was lowered. bFGF immunoreactivity was elevated in the INL and ONL in the PBS-injected eye (C) and the BMSC-transplanted eye (D), the latter of which experienced greater immunoreactivity. Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Figure 5.
 
Comparison of BDNF immunoreactivity of the S2–S3 retina among groups. Expression of BDNF in retina was identified by rabbit polyclonal anti–rat BDNF antibody. BDNF immunoreactivity (red) was detected by TRITC-labeled anti–rabbit antibody. BDNF immunoreactivity was located within cells in the GCL and INL in all eyes. There was no difference between the normal eye (A) and the light-damaged eye (B). There was similarly elevated BDNF immunoreactivity in the PBS-injected eye (C) and the BMSC-transplanted eye (D) compared with the normal eye and the light-damaged eye. Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Figure 5.
 
Comparison of BDNF immunoreactivity of the S2–S3 retina among groups. Expression of BDNF in retina was identified by rabbit polyclonal anti–rat BDNF antibody. BDNF immunoreactivity (red) was detected by TRITC-labeled anti–rabbit antibody. BDNF immunoreactivity was located within cells in the GCL and INL in all eyes. There was no difference between the normal eye (A) and the light-damaged eye (B). There was similarly elevated BDNF immunoreactivity in the PBS-injected eye (C) and the BMSC-transplanted eye (D) compared with the normal eye and the light-damaged eye. Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Figure 6.
 
Comparison of CNTF immunoreactivity of S2–S3 retina among groups. Expression of CNTF in retina was identified by mouse monoclonal anti–rat CNTF antibody. CNTF immunoreactivity (green) was detected by FITC-labeled anti–mouse antibody. CNTF immunoreactivity was faintly detected in the normal eye (A) but became intense in the INL and GCL in the light-damaged eye (B). There was lower CNTF immunoreactivity, primarily located in the GCL, in the PBS-injected eye (C) and the BMSC-transplanted eye (D) than in the light-damaged eye. Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Figure 6.
 
Comparison of CNTF immunoreactivity of S2–S3 retina among groups. Expression of CNTF in retina was identified by mouse monoclonal anti–rat CNTF antibody. CNTF immunoreactivity (green) was detected by FITC-labeled anti–mouse antibody. CNTF immunoreactivity was faintly detected in the normal eye (A) but became intense in the INL and GCL in the light-damaged eye (B). There was lower CNTF immunoreactivity, primarily located in the GCL, in the PBS-injected eye (C) and the BMSC-transplanted eye (D) than in the light-damaged eye. Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Integration and Differentiation of Transplanted BMSCs
Two weeks after transplantation, DAPI-positive cells were distributed in the injected area (the superior quadrant) and close to S2 to S3 retinas. Cells were distributed primarily in the subretinal space, and a few cells migrated to neural retinas. No transplanted DAPI-positive cells expressed GFAP, MAP2, nestin, rhodopsin, or calretinin. 
Cytokine Expression by Transplanted BMSCs
Immunohistochemistry against bFGF, CNTF, and BDNF was conducted on the retinas 2 weeks after transplantation. Most DAPI-positive BMSCs could express BDNF in the subretinal space and neural retinas but did not express bFGF or CNTF (Fig. 7). 
Figure 7.
 
Cytokine expression by transplanted DAPI-labeled BMSCs 2 weeks after transplantation. Expression of BDNF (A, B) and bFGF (C, D) by transplanted BMSCs (blue nuclei) were identified by rabbit polyclonal anti–rat BDNF and bFGF antibodies. Their immunoreactivity (red) was detected by TRITC-labeled anti–rabbit antibody. Expression of CNTF (E, F) by transplanted BMSCs was identified by mouse monoclonal anti–rat CNTF antibody. Its immunoreactivity (green) was detected by FITC-labeled anti–mouse antibody. (A) Some transplanted cells expressed BDNF in the ONL and the subretinal space. No transplanted cells expressed bFGF (C) or CNTF (E). (B, D, F) High-magnification images of insets in (A), (C), and (E), respectively. Scale bar, 100 μm.
Figure 7.
 
Cytokine expression by transplanted DAPI-labeled BMSCs 2 weeks after transplantation. Expression of BDNF (A, B) and bFGF (C, D) by transplanted BMSCs (blue nuclei) were identified by rabbit polyclonal anti–rat BDNF and bFGF antibodies. Their immunoreactivity (red) was detected by TRITC-labeled anti–rabbit antibody. Expression of CNTF (E, F) by transplanted BMSCs was identified by mouse monoclonal anti–rat CNTF antibody. Its immunoreactivity (green) was detected by FITC-labeled anti–mouse antibody. (A) Some transplanted cells expressed BDNF in the ONL and the subretinal space. No transplanted cells expressed bFGF (C) or CNTF (E). (B, D, F) High-magnification images of insets in (A), (C), and (E), respectively. Scale bar, 100 μm.
Differentiation and Cytokine Expression of BMSCs Cultivated by SHR
After 3 days, control cells became broad and flat, whereas approximately 10% of cells cultivated by SHR became multipolar and resembled neurocytes. Some processes connected flanking cells to each other (Fig. 8). When cultured for 1 day, some cells cultivated by SHR expressed nestin (positive rate: normal SHR, 36.7%; light-damaged SHR, 42.1%), but only a few cells (0.1%) in control medium expressed this. The positive rate of nestin gradually decreased with time, but some cells cultivated by light-damaged SHR began to express MAP2 (30.4%) or GFAP (57.8%) after 7 days (Fig. 9). Meanwhile, they expressed bFGF (66.7%) and BDNF (56.8%). None of the cells expressed rhodopsin or CNTF in vitro. 
Figure 8.
 
Morphologic changes of passage 2 BMSCs cultivated with SHR. (A) Cultivated in standard medium (DMEM-LG supplemented with 10% FBS), cells looked like fibroblasts. (B) Cultivated with only DMEM-LG, cells became broad and flat. (C) Cultivated with light-damaged SHR for 7 days, approximately 10% of cells changed into multipolar shape (red arrows). (D) Cultivated with light-damaged SHR for 7 days, processes of partial flanking cells connected. Scale bar, 100 μm.
Figure 8.
 
Morphologic changes of passage 2 BMSCs cultivated with SHR. (A) Cultivated in standard medium (DMEM-LG supplemented with 10% FBS), cells looked like fibroblasts. (B) Cultivated with only DMEM-LG, cells became broad and flat. (C) Cultivated with light-damaged SHR for 7 days, approximately 10% of cells changed into multipolar shape (red arrows). (D) Cultivated with light-damaged SHR for 7 days, processes of partial flanking cells connected. Scale bar, 100 μm.
Figure 9.
 
Differentiation of BMSCs cultivated with SHR. Expression of nestin was identified by mouse monoclonal anti–rat nestin antibody. Nestin immunoreactivity (green) was detected by FITC-labeled anti–mouse antibody. Expression of MAP2 and GFAP were identified by rabbit polyclonal anti–rat MAP2 and GFAP antibodies. MAP2 and GFAP immunoreactivities (red) were detected by TRITC-labeled anti–rabbit antibody. Cell nuclei (blue) were counterstained with Hoechst. (A) Cultivated with normal SHR for 1 day, 36.7% cells expressed nestin. (B) Higher magnification image of (A). (C) Cultivated with light-damaged SHR for 1 day, 42.1% of cells expressed nestin. (D) Higher magnification image of (C). Cultivated with light-damaged SHR for 7 days, 30.4% of cells expressed MAP2 (E), and 57.8% of cells expressed GFAP (F). Scale bar, 100 μm.
Figure 9.
 
Differentiation of BMSCs cultivated with SHR. Expression of nestin was identified by mouse monoclonal anti–rat nestin antibody. Nestin immunoreactivity (green) was detected by FITC-labeled anti–mouse antibody. Expression of MAP2 and GFAP were identified by rabbit polyclonal anti–rat MAP2 and GFAP antibodies. MAP2 and GFAP immunoreactivities (red) were detected by TRITC-labeled anti–rabbit antibody. Cell nuclei (blue) were counterstained with Hoechst. (A) Cultivated with normal SHR for 1 day, 36.7% cells expressed nestin. (B) Higher magnification image of (A). (C) Cultivated with light-damaged SHR for 1 day, 42.1% of cells expressed nestin. (D) Higher magnification image of (C). Cultivated with light-damaged SHR for 7 days, 30.4% of cells expressed MAP2 (E), and 57.8% of cells expressed GFAP (F). Scale bar, 100 μm.
Discussion
Retinal light damage can induce degeneration and apoptosis of photoreceptors, leading to irreversible vision impairment. However, there is still no direct way to prevent the death of photoreceptors. In this study, we attempted to transplant BMSCs into light-damaged rat eyes with the aim of compensating for the loss of photoreceptors. 
We used a direct adherence method to cultivate BMSCs. 11,24,25 Almost all second-passage cells expressed CD44, CD166 (the markers for stromal cells), and CD90 (an undifferentiated cell marker), and 0.2% cells expressed nestin (a neural stem cell marker) but never expressed CD34 3 (a surface marker for hematopoietic cells), MAP2 (a marker for mature neurons), GFAP (a marker for neuroglia), rhodopsin (a marker for photoreceptors), or calretinin 10,26 (a marker for amacrine cells), indicating that almost all the cultivated cells were undifferentiated stromal cells. 1 Similar to the findings of Garcia et al., 15 most of the cultivated second-passage BMSCs could express bFGF, CNTF, and BDNF, confirming that BMSCs had trophic potency in vitro. 
We chose the most severely affected location of light-damaged retinas (S2–S3) as the objects of comparison. Photoreceptor apoptosis was significantly reduced, and retinal structure was obviously improved in the BMSC transplantation group compared with the PBS group and the light damage group. The inhibition of photoreceptor apoptosis by BMSC subretinal transplantation has been reported in RCS rat 22,23 and rhodopsin knockout mouse 27 models, but it has never been reported in light-damaged rats. We also found that PBS injection could inhibit apoptosis to some extent, in accordance with the findings of Faktorovich et al. 28 Valter et al. 29 found that mechanical trauma to retinas could increase the expression of endogenous protective cytokines (CNTF and bFGF), which could inhibit apoptosis in light-damaged retinas. 
We transplanted BMSCs into the superior subretinal space, which was the nearest location to photoreceptors, consistent with previous studies. 22,23,27 Previous studies dealing with BMSC transplantation into the subretinal space of the RCS rat revealed that transplanted cells integrated into the ONL or retinal pigment epithelium layer adjacent to the injection site. In those cases, the engrafted cells expressed rhodopsin or cytokeratin, respectively. However, in our study, most of the BMSCs survived in the subretinal space 2 weeks after transplantation, and only a few integrated into the neural retina (most of them localized in the ONL). No engrafted cells differentiated into neural or retinal cells. There seemed to be three reasons for the lack of differentiation of BMSCs in our study. One was the short time of observation. Two weeks may be not enough for BMSCs to integrate and differentiate. Another was the difficulty of integrating engrafted BMSCs into the retina. Kicic et al. 10 found more BMSCs migrated into the ONL of RCS rats 2 weeks after subretinal transplantation than occurred our study. Perhaps our model did not facilitate BMSC integration into the retina. The final reason was that the microenvironment surrounding the engrafted cells was not sufficient for the cells to differentiate. Although a few studies demonstrated that engrafted cells expressed photoreceptor markers, 10,11 cells did not exhibit morphologic changes of outer and inner segment until 3 months after transplantation. Thus, differentiation was only partial and could not replace apoptotic photoreceptors morphologically and functionally. Adding specific exogenous factors or genetically modifying BMSCs might be considered to attain fully differentiated retinal cells. 
Inhibition of photoreceptor apoptosis by neurotrophic factors, such as CNTF, bFGF, and BDNF, has been reported in several previous studies. 2835 In our study, no transplanted BMSCs were observed to differentiate into neural or retinal cells to replace the lost photoreceptors; therefore, we suspected that the rescue effects observed after BMSC transplantation were the result of the trophic effects of BMSCs. By immunohistochemistry, the transplanted BMSCs showed BDNF expression, confirmed by in vitro culture with light-damaged SHR. Compared with the simple administration of BDNF, which has a short half-life, 36 engrafted BMSCs can survive for at least several months 37 and may continue to release BDNF. In addition, we observed elevated bFGF immunoreactivity in the S2–S3 retinas of BMSC-transplanted eyes. Although neither the origins nor the mechanisms of production of bFGF were clear, bFGF could be one of the reasons for rescue effects. Based on results indicating that BMSCs could express bFGF when cultured with light-damaged SHR for 7 days, we thought that the engrafted BMSCs could possibly express bFGF earlier (<2 weeks) to protect the retinas against light damage. 
In conclusion, this study showed that BMSC subretinal transplantation could inhibit photoreceptor apoptosis and slow down retinal damage in light-damaged rat eyes. Transplanted BMSCs survived in the subretinal space and ONL but did not differentiate into neural cells or retinal cells. BMSCs have the capacity to produce some kinds of trophic factors in the light-damaged model eye and in vitro cultivation with light-damaged SHR, which might have trophic and protective effects on light-damaged retina. Further study is needed to determine whether BMSCs can differentiate into photoreceptors in the light-damaged retina under different conditions and to find out the precise mechanisms by which BMSCs affect the light-damaged retina. This study strongly indicates that transplanted BMSCs are worthy of treatment of diseases associated with photoreceptor apoptosis. 
Footnotes
 Disclosure: Y. Zhang, None; W. Wang, None
References
Pittenger MF Mackay AM Beck SC . Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143–147. [CrossRef] [PubMed]
Seshi B Kumar S King D . Multilineage gene expression in human bone marrow stromal cells as evidenced by single-cell microarray analysis. Blood Cells Mol Dis. 2003;31:268–285. [CrossRef] [PubMed]
Sanchez-Ramos J Song S Cardozo-Pelaez F . Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol. 2000;164:247–256. [CrossRef] [PubMed]
Woodbury D Schwarz EJ Prockop DJ . Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res. 2000;61:364–370. [CrossRef] [PubMed]
Long X Olszewski M Huang W . Neural cell differentiation in vitro from adult human bone marrow mesenchymal stem cells. Stem Cells Dev. 2005;14:65–69. [CrossRef] [PubMed]
Tropel P Platet N Platel JC . Functional neuronal differentiation of bone marrow-derived mesenchymal stem cells. Stem Cells. 2006;24:2868–2876. [CrossRef] [PubMed]
Lei Z Yongda L Jun M . Culture and neural differentiation of rat bone marrow mesenchymal stem cells in vitro. Cell Biol Int. 2007;31:916–923. [CrossRef] [PubMed]
Chen J Li Y Wang L . Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke. 2001;32:1005–1010. [CrossRef] [PubMed]
Corti S Locatelli F Strazzer S . Modulated generation of neuronal cells from bone marrow by expansion and mobilization of circulating stem cells with in vivo cytokine treatment. Exp Neurol. 2002;177:443–452. [CrossRef] [PubMed]
Kicic A Shen WY Wilson AS . Differentiation of marrow stromal cells into photoreceptors in the rat eye. J Neurosci. 2003;23:7742–7749. [PubMed]
Timita M Adachi Y Yamada H . Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina. Stem Cells. 2002;20:279–283. [CrossRef] [PubMed]
Chiou SH Kao CL Peng CH . A novel in vitro retinal differentiation model by co-culturing adult human bone marrow stem cells with retinal pigmented epithelium cells. Biochem Biophys Res Commun. 2005;326:578–585. [CrossRef] [PubMed]
Tomita M Mori T Maruyama K . A comparison of neural differentiation and retinal transplantation with bone marrow-derived cells and retinal progenitor cells. Stem Cells. 2006;24:2270–2278. [CrossRef] [PubMed]
Chen X Li Y Wang L . Ischemic rat brain extracts induce human marrow stromal cell growth factor production. Neuropathology. 2002;22:275–279. [CrossRef] [PubMed]
Garcia R Aguiar J Alberci E . Bone marrow stromal cells produce nerve growth factor and glial cell line-derived neurotrophic factors. Biochem Biophys Res Commun. 2004;316:753–754. [CrossRef] [PubMed]
Sensebe L Deschaseaux M Li J . The broad spectrum of cytokine gene expression by myoid cells from the human marrow microenvironment. Stem Cells. 1997;15:133–143. [CrossRef] [PubMed]
Li Y Chen J Chen XG . Human marrow stromal cell therapy for stroke in rat: neurotrophins and functional recovery. Neurology. 2002;59:514–523. [CrossRef] [PubMed]
Horita Y Honmou O Harada K . Intravenous administration of glial cell line-derived neurotrophic factor gene-modified human mesenchymal stem cells protects against injury in a cerebral ischemia model in the adult rat. J Neurosci Res. 2006;84:1495–1504. [CrossRef] [PubMed]
Urdzíková L Jendelová P Glogarová K . Transplantation of bone marrow stem cells as well as mobilization by granulocyte-colony stimulating factor promotes recovery after spinal cord injury in rats. J Neurotrauma. 2006;23:1379–1391. [CrossRef] [PubMed]
Xiang P Huang HT Li HB . Effects of mesenchymal stem cells on regeneration of retinal ganglion cells in adult rats. Chin J Anat. 2005;28:252–254.
Yu S Tanabe T Dezawa M . Effects of bone marrow stromal cell injection in an experimental glaucoma model. Biochem Biophys Res Commun. 2006;344:1071–1079. [CrossRef] [PubMed]
Arnhold S Heiduschka P Klein H . Adenovirally transduced bone marrow stromal cells differentiate into pigment epithelial cells and induce rescue effects in RCS rats. Invest Ophthalmol Vis Sci. 2006;47:4121–4129. [CrossRef] [PubMed]
Inoue Y Iriyama A Ueno S . Subretinal transplantation of bone marrow mesenchymal stem cells delays retinal degeneration in the RCS rat model of retinal degeneration. Exp Eye Res. 2007;85:234–241. [CrossRef] [PubMed]
Meirelles Lda S Nardi NB . Murine marrow derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol. 2003;123:702–711. [CrossRef] [PubMed]
Rickard DJ Kassem M Hefferan TE . Isolation and characterization of osteoblast precursor cells from human bone marrow. J Bone Miner Res. 1996;11:312–324. [CrossRef] [PubMed]
Mendelow B Grobicki D Hunt M . Characterization of bone marrow stromal cells in suspension and monolayer cultures. Br J Haematol. 1980;46:15–22. [CrossRef] [PubMed]
Arnhold S Absenger Y Klein H . Transplantation of bone marrow-derived mesenchymal stem cells rescue photoreceptor cells in the dystrophic retina of the rhodopsin knockout mouse. Graefes Arch Clin Exp Ophthalmol. 2007;245:414–422. [CrossRef] [PubMed]
Faktorovich EG Steinberg RH Yasumura D . Basic fibroblast growth factor and local injury protect photoreceptors from light damage in the rat. J Neurosci. 1992;12: 3554–3567. [PubMed]
Valter K Bisti S Gargini C . Time course of neurotrophic factor upregulation and retinal protection against light induced damage after optic nerve section. Invest Ophthalmol Vis Sci. 2005;46:1748–1754. [CrossRef] [PubMed]
LaVail MM Unoki K Yasumura D . Multiple growth factors, cytokines, and neurotrophins rescue photoreceptors from the damaging effects of constant light. Proc Natl Acad Sci U S A. 1992;89:11249–11253. [CrossRef] [PubMed]
Gauthier R Joly S Pernet V . Brain-derived neurotrophic factor gene delivery to Müller glia preserves structure and function of light-damaged photoreceptors. Invest Ophthalmol Vis Sci. 2005;46:3383–3392. [CrossRef] [PubMed]
Masuda K Watanabe I Unoki K . Functional rescue of photoreceptors from the damaging effects of constant light by survival-promoting factors in the rat. Invest Ophthalmol Vis Sci. 1995;36:2142–2146. [PubMed]
Okoye G Zimmer J Sung J . Increased expression of brain-derived neurotrophic factor preserves retinal function and slows cell death from rhodopsin mutation or oxidative damage. J Neurosci. 2003;23:4164–4172. [PubMed]
Hojo M Abe T Sugano E . Photoreceptor protection by iris pigment epithelial transplantation transduced with AAV-mediated brain-derived neurotrophic factor gene. Invest Ophthalmol Vis Sci. 2004;45:3721–3726. [CrossRef] [PubMed]
Liu C Peng M Laties AM . Preconditioning with bright light evokes a protective response against light damage in the rat retina. J Neurosci. 1998;18:1337–1344. [PubMed]
Castren E Berninger B Leingartner A . Regulation of brain-derived neurotrophic factor mRNA levels in hippocampus by neuronal activity. Prog Brain Res. 1998;117:57–64. [PubMed]
Minamino K Adachi Y Yamada H . Long-term survival of bone marrow-derived retinal nerve cells in the retina. Neuroreport. 2005;16:1255–1259. [CrossRef] [PubMed]
Figure 1.
 
Characterization of P2-BMSCs by immunocytochemistry. Expression of (A) CD44, (B) CD166, (C) CD90, (D) Nestin, (E) CD11b, (F) bFGF, (G) CNTF, and (H) BDNF by BMSCs. CD44, CD166, and CD11b immunoreactivities (green) were detected by FITC-labeled secondary antibody, and the others immunoreactivities (red) were detected by TRITC-labeled secondary antibody. (AE) Cell nuclei (blue) were counterstained with Hoechst. Immunoreactivity of CD44, CD166, and CD90 was 98.5%, 95.6%, and 94.2%, respectively. 0.2% BMSCs expressed nestin and 1.5% BMSCs expressed CD11b. Scale bar, 100 μm.
Figure 1.
 
Characterization of P2-BMSCs by immunocytochemistry. Expression of (A) CD44, (B) CD166, (C) CD90, (D) Nestin, (E) CD11b, (F) bFGF, (G) CNTF, and (H) BDNF by BMSCs. CD44, CD166, and CD11b immunoreactivities (green) were detected by FITC-labeled secondary antibody, and the others immunoreactivities (red) were detected by TRITC-labeled secondary antibody. (AE) Cell nuclei (blue) were counterstained with Hoechst. Immunoreactivity of CD44, CD166, and CD90 was 98.5%, 95.6%, and 94.2%, respectively. 0.2% BMSCs expressed nestin and 1.5% BMSCs expressed CD11b. Scale bar, 100 μm.
Figure 2.
 
Comparison of S2–S3 retina structure among groups (hematoxylin and eosin stain). The number of ONL cells showed a significant difference between groups (P < 0.05, analysis of variance). (A) Normal eye. (B) In the light-damaged eye (24 days after light damage), the ONL cells decreased significantly. (C) The number of ONL cells was greater in the PBS-injected eye (24 days after light damage and 14 days after PBS injection) than in light-damaged eye. (D) The number of ONL cells was greater in the BMSC-transplanted eye (24 days after light damage and 14 days after BMSC transplantation) than in PBS injected eye. Scale bar, 100 μm. S2–S3 retina, retina 2 to 3 DD distance superior to the optic disc.
Figure 2.
 
Comparison of S2–S3 retina structure among groups (hematoxylin and eosin stain). The number of ONL cells showed a significant difference between groups (P < 0.05, analysis of variance). (A) Normal eye. (B) In the light-damaged eye (24 days after light damage), the ONL cells decreased significantly. (C) The number of ONL cells was greater in the PBS-injected eye (24 days after light damage and 14 days after PBS injection) than in light-damaged eye. (D) The number of ONL cells was greater in the BMSC-transplanted eye (24 days after light damage and 14 days after BMSC transplantation) than in PBS injected eye. Scale bar, 100 μm. S2–S3 retina, retina 2 to 3 DD distance superior to the optic disc.
Figure 3.
 
Comparison of apoptosis of S2–S3retina among groups. Apoptosis of ONL cells in each group was detected by TUNEL analysis (green) 24 days after light damage. Cell nuclei (blue) were counterstained with Hoechst. (A) Normal eye. (B) Light-damaged eye. (C) The percentage of apoptotic ONL cells was smaller in the PBS-injected eye (14 days after PBS injection) than in the light-damaged eye (P < 0.05). (D) The percentage of apoptotic ONL cells was smaller in the BMSC-transplanted eye (14 days after BMSC transplantation) than in the PBS-injected eye (P < 0.05), but the difference was not significant compared with the normal group (P > 0.05). Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Figure 3.
 
Comparison of apoptosis of S2–S3retina among groups. Apoptosis of ONL cells in each group was detected by TUNEL analysis (green) 24 days after light damage. Cell nuclei (blue) were counterstained with Hoechst. (A) Normal eye. (B) Light-damaged eye. (C) The percentage of apoptotic ONL cells was smaller in the PBS-injected eye (14 days after PBS injection) than in the light-damaged eye (P < 0.05). (D) The percentage of apoptotic ONL cells was smaller in the BMSC-transplanted eye (14 days after BMSC transplantation) than in the PBS-injected eye (P < 0.05), but the difference was not significant compared with the normal group (P > 0.05). Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Figure 4.
 
Comparison of bFGF immunoreactivity of S2–S3 retinas among groups. Expression of bFGF in retina was identified by rabbit polyclonal anti–rat bFGF antibody. BFGF immunoreactivity (red) was detected by TRITC-labeled anti–rabbit antibody. (A) In the normal eye, bFGF immunoreactivity was located in the INL. (B) In the light-damaged eye, bFGF was lowered. bFGF immunoreactivity was elevated in the INL and ONL in the PBS-injected eye (C) and the BMSC-transplanted eye (D), the latter of which experienced greater immunoreactivity. Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Figure 4.
 
Comparison of bFGF immunoreactivity of S2–S3 retinas among groups. Expression of bFGF in retina was identified by rabbit polyclonal anti–rat bFGF antibody. BFGF immunoreactivity (red) was detected by TRITC-labeled anti–rabbit antibody. (A) In the normal eye, bFGF immunoreactivity was located in the INL. (B) In the light-damaged eye, bFGF was lowered. bFGF immunoreactivity was elevated in the INL and ONL in the PBS-injected eye (C) and the BMSC-transplanted eye (D), the latter of which experienced greater immunoreactivity. Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Figure 5.
 
Comparison of BDNF immunoreactivity of the S2–S3 retina among groups. Expression of BDNF in retina was identified by rabbit polyclonal anti–rat BDNF antibody. BDNF immunoreactivity (red) was detected by TRITC-labeled anti–rabbit antibody. BDNF immunoreactivity was located within cells in the GCL and INL in all eyes. There was no difference between the normal eye (A) and the light-damaged eye (B). There was similarly elevated BDNF immunoreactivity in the PBS-injected eye (C) and the BMSC-transplanted eye (D) compared with the normal eye and the light-damaged eye. Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Figure 5.
 
Comparison of BDNF immunoreactivity of the S2–S3 retina among groups. Expression of BDNF in retina was identified by rabbit polyclonal anti–rat BDNF antibody. BDNF immunoreactivity (red) was detected by TRITC-labeled anti–rabbit antibody. BDNF immunoreactivity was located within cells in the GCL and INL in all eyes. There was no difference between the normal eye (A) and the light-damaged eye (B). There was similarly elevated BDNF immunoreactivity in the PBS-injected eye (C) and the BMSC-transplanted eye (D) compared with the normal eye and the light-damaged eye. Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Figure 6.
 
Comparison of CNTF immunoreactivity of S2–S3 retina among groups. Expression of CNTF in retina was identified by mouse monoclonal anti–rat CNTF antibody. CNTF immunoreactivity (green) was detected by FITC-labeled anti–mouse antibody. CNTF immunoreactivity was faintly detected in the normal eye (A) but became intense in the INL and GCL in the light-damaged eye (B). There was lower CNTF immunoreactivity, primarily located in the GCL, in the PBS-injected eye (C) and the BMSC-transplanted eye (D) than in the light-damaged eye. Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Figure 6.
 
Comparison of CNTF immunoreactivity of S2–S3 retina among groups. Expression of CNTF in retina was identified by mouse monoclonal anti–rat CNTF antibody. CNTF immunoreactivity (green) was detected by FITC-labeled anti–mouse antibody. CNTF immunoreactivity was faintly detected in the normal eye (A) but became intense in the INL and GCL in the light-damaged eye (B). There was lower CNTF immunoreactivity, primarily located in the GCL, in the PBS-injected eye (C) and the BMSC-transplanted eye (D) than in the light-damaged eye. Scale bar, 100 μm. S2–S3 retina, retina of 2 to 3 DD distance superior to the optic disc.
Figure 7.
 
Cytokine expression by transplanted DAPI-labeled BMSCs 2 weeks after transplantation. Expression of BDNF (A, B) and bFGF (C, D) by transplanted BMSCs (blue nuclei) were identified by rabbit polyclonal anti–rat BDNF and bFGF antibodies. Their immunoreactivity (red) was detected by TRITC-labeled anti–rabbit antibody. Expression of CNTF (E, F) by transplanted BMSCs was identified by mouse monoclonal anti–rat CNTF antibody. Its immunoreactivity (green) was detected by FITC-labeled anti–mouse antibody. (A) Some transplanted cells expressed BDNF in the ONL and the subretinal space. No transplanted cells expressed bFGF (C) or CNTF (E). (B, D, F) High-magnification images of insets in (A), (C), and (E), respectively. Scale bar, 100 μm.
Figure 7.
 
Cytokine expression by transplanted DAPI-labeled BMSCs 2 weeks after transplantation. Expression of BDNF (A, B) and bFGF (C, D) by transplanted BMSCs (blue nuclei) were identified by rabbit polyclonal anti–rat BDNF and bFGF antibodies. Their immunoreactivity (red) was detected by TRITC-labeled anti–rabbit antibody. Expression of CNTF (E, F) by transplanted BMSCs was identified by mouse monoclonal anti–rat CNTF antibody. Its immunoreactivity (green) was detected by FITC-labeled anti–mouse antibody. (A) Some transplanted cells expressed BDNF in the ONL and the subretinal space. No transplanted cells expressed bFGF (C) or CNTF (E). (B, D, F) High-magnification images of insets in (A), (C), and (E), respectively. Scale bar, 100 μm.
Figure 8.
 
Morphologic changes of passage 2 BMSCs cultivated with SHR. (A) Cultivated in standard medium (DMEM-LG supplemented with 10% FBS), cells looked like fibroblasts. (B) Cultivated with only DMEM-LG, cells became broad and flat. (C) Cultivated with light-damaged SHR for 7 days, approximately 10% of cells changed into multipolar shape (red arrows). (D) Cultivated with light-damaged SHR for 7 days, processes of partial flanking cells connected. Scale bar, 100 μm.
Figure 8.
 
Morphologic changes of passage 2 BMSCs cultivated with SHR. (A) Cultivated in standard medium (DMEM-LG supplemented with 10% FBS), cells looked like fibroblasts. (B) Cultivated with only DMEM-LG, cells became broad and flat. (C) Cultivated with light-damaged SHR for 7 days, approximately 10% of cells changed into multipolar shape (red arrows). (D) Cultivated with light-damaged SHR for 7 days, processes of partial flanking cells connected. Scale bar, 100 μm.
Figure 9.
 
Differentiation of BMSCs cultivated with SHR. Expression of nestin was identified by mouse monoclonal anti–rat nestin antibody. Nestin immunoreactivity (green) was detected by FITC-labeled anti–mouse antibody. Expression of MAP2 and GFAP were identified by rabbit polyclonal anti–rat MAP2 and GFAP antibodies. MAP2 and GFAP immunoreactivities (red) were detected by TRITC-labeled anti–rabbit antibody. Cell nuclei (blue) were counterstained with Hoechst. (A) Cultivated with normal SHR for 1 day, 36.7% cells expressed nestin. (B) Higher magnification image of (A). (C) Cultivated with light-damaged SHR for 1 day, 42.1% of cells expressed nestin. (D) Higher magnification image of (C). Cultivated with light-damaged SHR for 7 days, 30.4% of cells expressed MAP2 (E), and 57.8% of cells expressed GFAP (F). Scale bar, 100 μm.
Figure 9.
 
Differentiation of BMSCs cultivated with SHR. Expression of nestin was identified by mouse monoclonal anti–rat nestin antibody. Nestin immunoreactivity (green) was detected by FITC-labeled anti–mouse antibody. Expression of MAP2 and GFAP were identified by rabbit polyclonal anti–rat MAP2 and GFAP antibodies. MAP2 and GFAP immunoreactivities (red) were detected by TRITC-labeled anti–rabbit antibody. Cell nuclei (blue) were counterstained with Hoechst. (A) Cultivated with normal SHR for 1 day, 36.7% cells expressed nestin. (B) Higher magnification image of (A). (C) Cultivated with light-damaged SHR for 1 day, 42.1% of cells expressed nestin. (D) Higher magnification image of (C). Cultivated with light-damaged SHR for 7 days, 30.4% of cells expressed MAP2 (E), and 57.8% of cells expressed GFAP (F). Scale bar, 100 μm.
Table 1.
 
Primary Antibodies in Immunocytochemistry to Characterize BMSCs
Table 1.
 
Primary Antibodies in Immunocytochemistry to Characterize BMSCs
Primary Antibody Host Titer Cell Supplier
Anti-CD34 Mouse 1:100 Hematopoietic cell Santa Cruz Biotechnology (Santa Cruz, CA)
Anti-CD90 Mouse 1:500 Undifferentiated cell Chemicon (Temecula, CA)
Anti-CD44 Goat 1:100 Stromal cell Santa Cruz Biotechnology
Anti-CD166 Goat 1:100 Stromal cell Santa Cruz Biotechnology
Anti-nestin Mouse 1:50 Neural stem cell Chemicon
Anti-MAP2 Rabbit 1:400 Neuron Chemicon
Anti-rhodopsin Mouse 1:200 Rod photoreceptor Chemicon
Anti-calretinin Mouse 1:1500 Amacrine cell Chemicon
Anti-GFAP Rabbit 1:400 Neuroglial cell Chemicon
Anti-CD11b Mouse 1:50 Phagocyte Chemicon
Anti-bFGF Rabbit 1:1000 Chemicon
Anti-BDNF Rabbit 1:1000 Chemicon
Anti-CNTF Mouse 1:50 Chemicon
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