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Keywords:

  • Tissue regeneration;
  • Bone marrow mononuclear cells;
  • Mesenchymal stem cells;
  • Glaucoma;
  • Intraocular pressure

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Among bone marrow cells, hematopoietic and mesenchymal components can contribute to repair damaged organs. Such cells are usually used in acute diseases but few options are available for the treatment of chronic disorders. In this study, we have used a laser-induced model of open angle glaucoma (OAG) to evaluate the potential of bone marrow cell populations and the mechanisms involved in tissue repair. In addition, we investigated laser-induced tissue remodeling as a method of targeting effector cells into damaged tissues. We demonstrate that among bone marrow cells, mesenchymal stem cells (MSC) induce trabecular meshwork regeneration. MSC injection into the ocular anterior chamber leads to far more efficient decrease in intraocular pressure (IOP) (p < .001) and healing than hematopoietic cells. This robust effect was attributable to paracrine factors from stressed MSC, as injection of conditioned medium from MSC exposed to low but not to normal oxygen levels resulted in an immediate decrease in IOP. Moreover, MSC and their secreted factors induced reactivation of a progenitor cell pool found in the ciliary body and increased cellular proliferation. Proliferating cells were observed within the chamber angle for at least 1 month. Laser-induced remodeling was able to target MSC to damaged areas with ensuing specific increases in ocular progenitor cells. Thus, our results identify MSC and their secretum as crucial mediators of tissue repair in OAG through reactivation of local neural progenitors. In addition, laser treatment could represent an appealing strategy to promote MSC-mediated progenitor cell recruitment and tissue repair in chronic diseases. STEM Cells 2013;31:1136–1148


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

One of the most important breakthroughs of the last decade is the discovery that stem cells from various tissues have the ability to repair damaged organs. However, the repair mechanisms used by such cells are only beginning to be elucidated. Such effects can be mediated by undifferentiated stem cells transforming into mature cells with specialized function [1–4]. Alternatively, stem cells could also secrete cytokines and other factors that enhance intrinsic repair mechanisms and promote the development of a protective environment capable of changing the outcome for cells that have sustained potentially lethal damage [5–10]. Experimental data support immune modulation and neovascularization as mechanisms, but few other pathways have been thoroughly investigated [11–14]. In addition, it has been shown that stem cells are particularly active when injected soon after the initiation of acute tissue damage or in the presence of active disease [10, 15, 16]. In chronic disorders or instances where scarring has already developed, decreased homing signals and release of cytokines within dysfunctional or fibrotic tissues may limit the efficacy of cellular interventions. Thus, there is a dire need to develop approaches to create space and enhance stem cell recruitment to such tissues.

Among the various organs targeted by cellular interventions, the eye offers particularly attractive features to rapidly advance cell therapy [17]. Indeed the eye is immediately accessible for cellular injection and allows rapid as well as direct visualization and monitoring of the impact of therapeutic interventions. One of the most important chronic optic neuropathies and an important cause of blindness worldwide is open angle glaucoma (OAG) [18]. It is primarily associated with increased resistance to the outflow of aqueous humor (AH) through the trabecular meshwork (TM) resulting from apoptotic cell loss and subsequent increase in extracellular matrix density [19–25]. Elevated intraocular pressure (IOP) is the most important risk factor for glaucoma, as changes in IOP homeostasis can result in damage to the optic nerve, and reduction of IOP is the mainstay of therapy [26, 27]. Treatments include life-long medication to lower the production of AH, laser treatment of the TM to temporarily help regulate IOP, or invasive surgical procedures to allow AH outflow through alternate routes [28–31]. With the possible exception of laser trabeculoplasty, current procedures do not attempt to treat the TM. Given that TM degeneration is one of the root causes of IOP elevation in OAG, the development of an efficient way to regenerate the TM could improve IOP control and potentially treat many patients with this disease.

In this study, we investigated bone marrow (BM) cell injection to promote TM regeneration and reduce the elevated IOP in an animal glaucoma model. We showed that among BM cells, mesenchymal stem cells (MSC) migrated predominantly to the area of tissue damage and were particularly efficient at decreasing IOP, restoring AH drainage, and causing histological tissue repair. Such favorable results were observed although MSC persisted for only a few days, and their effects were replicated through local injection of their secretum. In addition, MSC promoted the reactivation and proliferation of nestin-expressing ocular progenitor cells in the ciliary body which, in turn, persisted in the eye for the duration of the study. Moreover, laser therapy led to both effector cell homing and localized recruitment of endogenous nestin expressing progenitors, thereby opening the door to targeted cell-based therapy of chronic ocular and other disorders.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Animals

Six to eight week old C57BL/6 male mice and 3-month-old Brown Norway female rats were purchased from Jackson Laboratories (Bar Harbor, ME, http://www.jax.org) and Charles River (Kingston, ON, Canada, http://www.criver.com), respectively. Animals were housed in pathogen-free conditions and studied using protocols approved by the Hôpital Maisonneuve-Rosemont (HMR) Research Center Animal Protection Committee.

Isolation, Culture, and Expansion of BM Mononuclear Cells

BM was aspirated from femurs and tibias of 2-month-old C57BL/6 mice. BM mononuclear cells (BMMCs) were isolated by using density gradient centrifugation (Ficoll-Paque; Amersham Pharmacia Biotech, Montreal, QC, Canada, http://www.amersham.com) and suspended in Dulbecco's modified Eagle's medium (DMEM; Gibco, Burlington, ON, Canada, http://www.invitrogen.com), 20% heat-inactivated (56°C, 60 minutes) fetal bovine serum (FBSI; Hyclone, Logan, UT http://www.hyclone.com), 2 μM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). Cells were plated in 75 cm2 tissue culture flasks (Sarstedt, Montreal, QC, Canada, http://www.sarstedt.com) and incubated at 37°C in 5% CO2 humidified air. After 48 hours, nonadherent cells were removed, grown until approximately 90% confluence, and then passaged with medium changes every 3–4 days.

MSC Characterization

After the second passage, BMMC were characterized for mesenchymal and hematopoietic markers by flow cytometry analysis. The following fluorochrome-labeled anti-mouse antibodies were used: CD14- and CD45-Allophycocyanin (APC), CD105-Pacific blue, CD73-Phycoerythrin (PE) (all eBioscience, San Diego, CA, http://www.ebioscience.com), and CD90-fluorescein isothiocyanate (FITC) (BD, Mississauga, ON, Canada, http://www.bd.com). Data were collected on a FACS LSRII cytometer (BD) and analysis was performed with FlowJo software (Tree Star, Ashland, OR, http://www.treestar.com). Cells positive for the hematopoietic marker CD45 were sorted using a FACS Aria III (BD), and both MSC and hematopoietic cells were used for in vivo experiments. MSC were tested for osteogenic and adipogenic differentiation using osteocyte/chondrocyte or adipocyte differentiation media (Invitrogen, Burlington, ON, Canada, http://www.invitrogen.com), and alizarin red S and oil red O staining (Sigma, Oakville, ON, Canada, http://www.sigmaaldrich.com), respectively.

TM Cell Culture

The TM cell line MUTM-NEI/1 originating from H2Kb-tsA48 heterozygote mice (genetic background CBA/ca X C57BL/10) was a kind gift of Dr. Joram Piatigorsky [32]. Cells were cultured in nonpermissive conditions (37°C) in 75 cm2 adherent tissue culture flasks with TM cell medium consisting of Iscove's Modified Dulbecco's Medium (Gibco) supplemented with 20% FBSI, and L-glutamine, penicillin, and streptomycin at above concentrations.

Coculture Condition

MSC to TM cell differentiation was assessed in coculture experiments. Briefly, 1 × 106 MSC were seeded in a 75 cm2 tissue culture flask and stained using CellTracker Blue CMAC according to manufacturer's protocol (Invitrogen). Cells were washed with Hanks' medium before adding fresh TM cell culture medium and seeding of 1 × 106 MUTM-NEI/1 cells at day 0 of coculture. After 7 days, cells were harvested and expression of TM cell markers was analyzed using flow cytometry. The following unlabeled antibodies were used according to manufacturer's protocol: rabbit anti-mouse Pax6, aquaporin-1 (both from US Biological, Burlington, ON, Canada, http://www.usbio.net), laminin (Sigma), and fibronectin (Millipore, Nepean, ON, Canada, http://www.millipore.com). Primary antibodies were revealed with goat anti-rabbit AlexaFluor488 (Invitrogen).

MSC Culture Medium for Paracrine Studies

MSC secretum was obtained after seeding 1 × 106 MSC in 15 mL DMEM for 24 hours at 37°C in 5% CO2 humidified air at either 21% or 5% O2. MSC conditioned medium (CM) was then harvested and concentrated 40× using 10 kDa centrifugal filter devices (Amicon Ultra-15, Millipore).

Glaucoma Model

To induce ocular hypertension, rats were anesthetized using intraperitoneal injection of ketamine (50 mg/kg; Wyeth, Guelph, ON, Canada, http://www.pfizer.com) and xylazine (2.2 mg/kg; Bayer, Toronto, ON, Canada, http://www.bayer.com), and drops of the local anesthetic proparacaine (Alcon Canada, Mississauga, ON, Canada, http://www.alcon.ca) were applied to the eyes. The method developed by Levkovitch-Verbin et al. [33]. was modified to selectively damage only half of the circumference of the anterior chamber angle Briefly, 90 spots of 50 μm diameter were directed toward the nasal 180° of the TM area using a Coherent Novus Omni laser (Laser Labs; Tampa, FL, http://www.laserlabs.com) set at 532 nm wavelength, 200 mW, and 0.1-second duration. Baseline IOP was measured weekly with a Tonopen-XL (Medtronic, Mississauga, ON, Canada, http://www.medtronic.ca). IOP was obtained in rats by performing an average of 10 measurements at a time by a blinded observer.

Transplantation

Before injection, cells were stained using carboxyfluorescein succinimidyl ester (CFSE, CellTrace, Invitrogen) dye. A control population was kept in vitro in BMMC medium to measure fluorescence changes over time. CFSE-stained cells were suspended at 1 × 105 cells per μL in saline solution (Baxter, Pointe-Claire, QC, Canada, http://www.baxter.ca). Intraocular transplantation was performed during the hour or 4 days following laser treatment. Prior to intraocular injection, 10 μL of AH was removed from the anterior chamber of the treated eye using a 281/2 gauge needle under direct observation with a binocular surgical microscope. Injection of 10 μL of cell preparation (cells, saline solution-placebo, or MSC-CM) was performed at the same injection site immediately after AH removal and under identical conditions. Injection was carried out over the course of 1 minute to avoid additional stress to the anterior chamber. Although minimal reflux was observed after injection, no leaks were observed beyond the first 24 hours when using the Seidel test. Rare animals (two for the duration of the study) have sustained additional damage to the anterior chamber during the injection and were removed from the experiment. All animals were kept under immunosuppressed conditions by daily intraperitoneal injection of cyclosporine A (Sandimmune; Novartis Pharma, Montreal, QC, Canada, http://www.novartis.com) starting 3 days before treatment and continued for the duration of the experiment.

Tissue Processing

Animals were euthanized by intraperitoneal injection of pentobarbital sodium (Euthanyl; Bimeda-MTC Animal Health, Cambridge, ON, Canada, http://www.bimedamtc.comy). Enucleated eyes were embedded in Optimum Cutting Temperature (Tissue-tek O.C.T.; Fisher Scientific, Ottawa, ON, Canada, http://www.fisherscientific.com) compound and flash-frozen in liquid nitrogen. Twelve micrometer sections were cut by cryostat (Leica, Richmond Hill, ON, Canada, http://www.leica.com) and mounted on slides (Fisher Scientific). After fixation in 4% paraformaldehyde, hematoxylin and eosin staining (Sigma) was used to examine anterior segment histology.

AH Outflow

To monitor AH outflow, we relied on a modified methodology from Gabelt et al. [34]. Briefly, 5 μL of C-14 labeled dextran (Dextran [carboxyl-14C] Avg M.W. 70,000, American Radiolabeled Chemicals, St-Louis, MO, http://www.arc-inc.com) was injected on day 8 over the course of 1 minute in the anterior chamber of rats from laser-treated, laser + low oxygen CM-treated, or untreated groups (n = 3 per group). One hour prior to intraocular injection, unlabeled dextran (0.5 mL, 100 mg/mL, Sigma) was injected intravenously to reduce risk of radiolabeled dextran entrapment in organs. To calculate trabecular outflow, blood samples were taken from a cannula implanted in the jugular vein on the side of the treated eye of anesthetized animals. Blood samples were collected every 2 minutes after dextran C-14 injection to monitor re-entry of the radiolabeled marker to the blood.

EDU Labeling

To monitor the fate of anterior chamber cells during tissue regeneration, nine animals were injected intraperitoneally with 50 mg/kg of 5-ethynyl-2'-deoxyuridine (EDU) (Invitrogen) from days 1 to 3 after laser damage and intraocular injections [35].

Immunofluorescence

Samples were incubated for 1 hour with blocking-permeabilizing solution of phosphate buffered saline (PBS) containing 1 mg/ml bovine serum albumin (Vector Labs., Burlington, ON, Canada, http://www.vectorlabs.com) and 1 μL/mL Tween (Sigma). Slides were incubated overnight at 4°C with unlabeled mouse anti-nestin, rabbit anti-Ki67 (BD), and rabbit anti F4-80 antibodies (Abcam, Toronto, ON, Canada, http://www.abcam.com). Primary antibodies were revealed with goat anti-rabbit AlexaFluor488 and goat anti-mouse AlexaFluor594 (all from Invitrogen). Nuclei were stained with mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (Vectashield; Vector Labs). EDU labeling was performed with Click-it EDU Alexa Fluor 488 imaging kit (Invitrogen) according to manufacturer's instructions. Histological sections were observed by confocal microscopy (Axiovert 100 LSM 510Meta; Carl Zeiss, Jena, Germany, http://www.zeiss.com) while fluorescence and surface area of anterior chamber structures were analyzed using AxioVs40 V4.8.2.0 (Carl Zeiss).

TUNEL Assay

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed using the In Situ Cell Death Detection Kit, Fluorescein (Roche, Basel, Switzerland, http://www.roche-applied-science.com) according to the manufacturer's instructions. Briefly, ocular sections were permeabilized in 0.1% Tween for 15 minutes. Sections were then incubated with the enzyme terminal deoxynucleotidyl transferase and fluorescein-conjugated dUTP at 37°C for 1 hour. Nuclear DNA was stained with DAPI (Vector Labs).

Statistical Analysis

Comparisons between animal groups were done using two-way ANOVA and Mann–Whitney t test. Statistical analyses were performed using GraphPad Prism version 4.0 (San Diego, CA, http://www.graphpad.com). All statistical tests were two-sided and performed at the significance level of .05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Animal Glaucoma Model

In order to study the contribution of MSC to the regeneration of the TM in glaucoma, we opted for a modified version of the laser-induced animal model developed by Levkovitch-Verbin et al. which allowed for an internal control consisting of an untreated segment within the laser-treated eye. Laser treatment covering 180° of the anterior chamber angle resulted in a significant elevation of the IOP, with a peak IOP of 26.9 ± 0.7 mmHg at day 12 (Fig. 1A, mean ± SEM; n = 10; p < .001), as opposed to healthy control animals with peak IOP of 20.8 ± 0.2 mmHg (Fig. 1A, n = 10). Laser-induced IOP elevation persisted for a period of approximately 30 days.

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Figure 1. MSC induce a rapid return to normal IOP levels in experimental glaucoma. (A): Ocular anterior chambers were either injected with 1 × 106 BMMC (red), 1 × 106 lymphocytes (black), saline (green), or received no additional treatment (blue). The gray area represents IOP normal range. IOP was reported as mean ± SEM of four experiments evaluating 12 animals per group. (B): 0.5 × 106 CD45 + cells (black) or 0.5 × 106 MSC (red line) were injected intraocularly after laser exposure and evaluated as described above. Mean ± SEM of three experiments evaluating nine animals per group. (C): Cumulative IOP exposure in eyes that received laser damage and injection of different cellular populations and controls. Calculated as the integral of IOP over the 4-week experimental period. (D): TM cross-section area measurements in μm2. TM surface area was measured for each experimental control (red, laser + MSC group; green, laser + lymphocyte group) and compared to the surface area of a normal TM (gray) (E): Representative images of hematoxylin-eosin (H&E) staining of rat ocular anterior segment before (C: control) and 1 week after laser exposure alone or followed by MSC injection, as well as 4 weeks after laser damage alone or followed by MSC injection. Significance compared to laser control group: *, p < .05; **, p < .01; ***, p < .001. Scale bar = 50 μm. Abbreviations: BMMCs, bone marrow mesenchymal stem cells; CB, ciliary body; Co, cornea; IOP, intraocular pressure; I, iris; MSC, mesenchymal stem cells; TM, trabecular meshwork.

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Injection of BM Cells In Vivo Decreases IOP

We first investigated the ability of cultured BMMCs to promote IOP pressure recovery in vivo (Fig. 1A). Increased IOP was observed in all groups on day 2 after laser exposure. While IOP continued to rise in laser-treated animals, peaking on day 10, the ocular injection of 1 × 106 BMMCs rapidly lowered IOP, which returned to baseline significantly earlier (day 8) than in the laser only group (day 32; p < .01). Injection of saline solution resulted in IOP similar to untreated controls. Cellular controls, which consisted of lymphocytes, failed to impact on IOP curves. The nature of injection content had no effect on IOP levels at day 2 for all groups (mean ranging from 26.5 to 27.7 in all four groups). However, the time required for BMMC to reduce the mean IOP by 50% was 4.3 days, five times faster than the time required to obtain the same pressure drop in the laser only (20.5 days), saline (22.5 days), or lymphocyte (22.2 days; p < .001) control groups.

BMMC Characterization

Adherent BMMCs harvested from B6 mice and expanded in culture in vitro were tested for hematopoietic markers (CD45 and CD14). After two passages, 65% of these adherent cells were negative for CD45 and CD14 (Supporting Information Fig. 1A). These cells expressed classical MSC markers: CD73, CD90, and CD105 (Supporting Information Fig. 1B–1D) and were capable of adipogenic and osteogenic differentiation in the proper media, thus satisfying MSC criteria (Supporting Information Fig. 1E, 1F). To determine which of hematopoietic cells [36] or MSC mediated the IOP drop, BMMC were sorted according to CD45 expression and injected in vivo (Fig. 1B). Both hematopoietic (CD45 +) and MSC groups were injected the same number of cells (5 × 105 cells) corresponding approximately to that found in the total BMMC population. Laser only animals had a peak IOP of 26.8 ± 0.7 mmHg on day 6 and those injected with CD45 + cells peaked at 25.6 ± 0.6 mmHg on day 12. In contrast, the MSC group demonstrated a rapid fall in IOP on day 5 (21.6 ± 0.7 mmHg; n = 9; p < .01). The time required to reduce the pressure by 50% was 4.5 days, six times faster than the time required to obtain the same drop in the laser control (27.8 days) or CD45 + cells (26 days; p < .001). Additionally, cumulative IOP was significantly lower in groups treated with MSC (p < .01 for BMMC and p < .001 for MSC) than in the other injection control groups (Fig. 1C). Thus, these results indicate that within BMMCs, MSC are primarily responsible for the rapid restoration of baseline IOP.

Injection of MSC In Vivo Restores TM Structure

Ocular tissue sections were performed to investigate mechanisms used by MSC to promote healing of anterior chamber angle structures. One week postphotocoagulation, anterior chamber angle structures (iris, ciliary body, and TM) of damaged eyes showed signs of inflammation, desquamation, and anterior synechiae (Fig. 1E). Cellular composition of these structures at 1 week depended on the treatment, as pigmented cells were the dominant cell population in the laser only groups, with the additional presence of nonpigmented cells in the MSC-injected laser-treated eyes. One month after photocoagulation, eyes that did not receive MSC transplantation presented scarring in the TM area, as well as invasion by pigmented epithelial cells. In contrast, eyes that received a MSC graft had an angle structure and histology that approximated that of the intact eye. These observations were confirmed by measuring the surface area of the TM area (Fig. 1D). After 30 days, no significant changes could be observed between the normal TM surface area and the group that received MSC transplantation, while the group that did not received MSC had a significantly smaller TM surface (p < .01). These results indicate that MSC injection leads to efficient structural recovery of the anterior chamber angle.

MSC Do Not Transdifferentiate into TM Cells In Vitro

To investigate potential differentiation of MSC into TM cells upon exposure to TM cell environment, these two populations were cocultured in vitro. MSC could be tracked using fluorescent dye labeling (CellTracker Blue) and evaluated for the acquisition of TM features by monitoring the surface antigens aquaporin-1, fibronectin, and laminin as well as the nuclear transcription factor Pax6, since these molecules ensure the proper development and function of TM cells (Supporting Information Fig. 2). All MUTM-NEI/1 TM cells expressed these surface antigens and transcription factor. However, few MSC expressed laminin and aquaporin-1 in normal culture conditions. Expression levels of Pax6 and fibronectin were not found to be significantly different between both cell populations prior to coculture. After 7 days of coincubation, the MSC population failed to acquire expression of aquaporin and laminin indicating that direct contact with TM cells did not promote MSC differentiation.

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Figure 2. In vivo MSCs migration and survival. CFSE-stained MSC were injected in the anterior chamber and tracked in vivo. (A–F): Representative images of the intact (A, C, E) and laser-damaged (B, D, F) chamber angles are shown at 24, 48, and 96 hours, respectively, after laser damage and cell injection. Nuclei were DAPI stained. (G): Compilation of total number of CFSE + cells present in the eye at 24, 48, and 96 hours after cell injection. Cell numbers (mean ± SEM) were determined by fluorescence microscopy analysis of sequential ocular sections (n = 5 per time point). Percentages of residual cells are indicated according to the number of injected MSC. (H): Distribution of CFSE + cells to the intact or damaged area was measured at each time point. Arrow: TM. Significance: ***, p < .001. Scale bar = 50 μm. Abbreviations: AC, anterior chamber; Cb, cilliary body; Co, cornea; CFSE, carboxyfluorescein succinimidyl ester; DAPI, 4',6-diamidino-2-phenylindole; I, Iris; L, lens; MSC, mesenchymal stem cell.

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In Vivo MSCs Migration and Survival

To investigate the mechanisms implicated in IOP lowering, we evaluated the ability of MSC to migrate and integrate in vivo into the TM. CFSE labeling of MSC on the day of injection enabled their subsequent detection by immunofluorescence. Tissue sections from animals sacrificed at 24, 48, and 96 hours revealed a major decrease in the number of CFSE + cells over time (Fig. 2). Cells were no longer detectable in the anterior chamber 96 hours after their injection. MSC clearance may be due to ocular microglia phagocytosis, as approximately one in five CFSE + cells coexpressed microglia marker F 4/80 on day 2 (Supporting Information Fig. 3). CFSE expression of MSC kept in vitro in culture medium was detectable by fluorescent microscopy for 7 days (data not shown). Interestingly, 24 hours after MSC administration, a high number (2.2 ± 0.1 × 104) of cells had migrated to the area of laser damage while only half that number (1.2 ± 0.2 × 104; p < .01) was found in the intact area. Although this proportion was maintained at 48 hours (1.0 ± 0.2 × 104and 0.5 ± 0.2 × 104 in the damaged and intact areas, respectively) the difference was no longer found to be significant at that time. These findings demonstrate that MSC have the ability to specifically migrate to the area of laser damage but do not remain in the anterior chamber for a prolonged period of time.

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Figure 3. MSC secreted factors promote ocular regeneration. IOP was measured in animals exposed to laser treatment alone (blue) and with injection of MSC (red), saline solution (orange), or MSC-CM generated in normoxic (black) or low-oxygen (green) conditions. MSC-CM was injected either (A) immediately (day 0) or (B) 4 days after laser damage. The gray area represents the normal range of IOP. Black vertical lines correspond to the time of laser treatment, and black arrowhead to the time of injection of MSC or their CM. Results expressed as mean ± SEM of three independent experiments, each with three animals per group. (C): Cumulative IOP exposure in eyes that received laser damage and injection of different cellular populations and controls. Calculated as the integral of IOP over the 3-week experimental period. (D): TM cross-section area measurements in μm2. TM surface area was measured for each experimental control (red, laser + MSC group; green, laser + lymphocyte group) and compared to the surface area of a normal TM (gray) (E): Anterior ocular segments from rats treated immediately following laser exposure with normoxic or low-oxygen MSC-CM were stained with hematoxylin-eosin (H&E). Evaluations were performed at day 4 and 14 after MSC-CM injection. Significance compared to laser control group: *, p < .05; **, p < .01; ***, p < .001. Scale bar = 50 μm. Abbreviations: Cb, ciliary body; Co, cornea; CM, conditioned medium; I, Iris; IOP, intraocular pressure; MSC mesenchymal stem cell; TM, trabecular meshwork.

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MSC Secreted Factors Influence Ocular Regeneration

TM regeneration was observed although MSC rapidly cleared out from the chamber angle (96 hours), suggesting that these cells are not inducing tissue repair by integrating the TM and directly replacing the damaged cells. This could imply that their effect was instead mediated by secreted factors and by activation of an endogenous repair mechanism. Hypoxia and cytokines produced by immune cells or environing tissue are known to induce changes in MSC secreted factors [37–39]. To determine the implication of such a mechanism, MSC-CM was harvested after 24 hours in vitro culture under either normoxic or low-oxygen conditions. No significant effect on IOP was observed when laser-treated animals were injected concentrated (40×) normoxic MSC-CM (Fig. 3A). Interestingly, when concentrated (40×) low-oxygen CM was injected immediately after laser treatment, peak IOP and subsequent measurements were found to be similar to that of the MSC-treated group. We tested the robustness of our treatments by delaying the injection of low-oxygen CM, saline solution control, and MSC until 4 days after laser exposure to allow damage and inflammation to develop further in the anterior chamber (Fig. 3B). Again MSC and low-oxygen CM groups induced similar decreases in IOP while saline solution induced no significant improvements. Treatment on day 0 instead of day 4 resulted in lower cumulative IOP for both MSC and low-oxygen MSC-CM (p < .05; Fig. 3C). Histological sections revealed that 4 days after CM injection, anterior chamber angle tissue structures (iris, ciliary body, and TM) showed clear signs of inflammation, desquamation, and anterior synechiae (Fig. 3E) Two weeks after photocoagulation, eyes that received normoxic CM treatment still showed considerable signs of scarring. In contrast, eyes that received low-oxygen CM treatment had an angle structure and histology closer to that of the intact eye. These observations were confirmed by measurement of the TM surface area (Fig. 3D). After 2 weeks, no significant changes could be observed between the normal TM surface area and the group that received low-oxygen CM treatment while the group that received normoxic CM treatment had a significantly smaller TM surface (p < .01).

Additionally, injection of low-oxygen CM restored TM functionality by increasing AH outflow (Supporting Information Fig. 4). Indeed, clearance of the radiolabeled tracer was very low after laser damage when compared to normal controls with an estimated peak clearance of 0.04 ± 0.006 μL/minute versus 0.20 ± 0.06 μL/minute, respectively (p < .05). Low oxygen-CM groups demonstrated significantly greater humor clearance (0.11 ± 0.003 μL/minute, p < .05) than laser-treated groups. Overall, these results indicate that cellular stress is required for MSC to secrete therapeutic factors, and that such factors exert a key role in the functional recovery of ocular tissue.

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Figure 4. Mesenchymal stem cell (MSC) trigger appearance of nestin + cells. (A–C): Light microscopy and immunofluorescence images of trabecular meshwork (TM) area from ocular sections of laser-treated animals injected with (A) lymphocyte controls, (B) 0.5 × 106 MSC, or (C) low-oxygen MSC-CM and evaluated for stem/progenitor cells using nestin. Nestin + cells were identified at day 2 after laser treatment and colocalized with pigmented (darkened areas under light microscopy) and nonpigmented cells. (D): CFSE-stained MSC (green) injected at day 0 in laser-treated animals were evaluated on day 2 to measure coexpression of nestin (red) with and without DAPI nuclear staining (blue). Scale bar = 50 μm. Abbreviations: CFSE, carboxyfluorescein succinimidyl ester; DAPI, 4',6-diamidino-2-phenylindole.

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Decreased IOP Allows for Better Survival of Retinal Cells

To confirm the importance of decreased IOP for the survival of retinal cells, a TUNEL assay was performed on eyes treated with low-oxygen and normoxic CM (Supporting Information Fig. 5). After 12 days of sustained IOP, TUNEL-positive apoptotic cells were observed within the retina of all treated eyes but in significantly lower numbers in group treated with low-oxygen CM. These data indicate the importance of IOP drop in retinal cell survival.

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Figure 5. Nestin expression and cellular proliferation correlates with ocular regeneration. Graphs represent compilation of nestin and Ki67 expressing cells in both the intact and damaged chamber angles based on the treatment received: (A) laser-treated eye with lymphocyte control injection, (C) laser-treated eye with MSC injection, (E) laser-treated eye with normoxic CM injection, (G) laser-treated eye with low-oxygen CM injection. Three animals per group from two separate experiments (n = 6). Numbers indicate the percentage of positive cells found in the area composed of the ciliary body, the TM, and a limited portion of the iris. Immunofluorescence histological sections from a rat eye were stained for nestin and Ki67 (B, D, F, H). Pictures are representative images of each group at the time point of peak nestin expression. Nestin (red) Ki67 (green) and DAPI (blue). Significance comparing damaged to intact areas: *, p < .05; ***, p < .001. Scale bar = 100 μm. Abbreviations: CM, conditioned media; DAPI, 4',6-diamidino-2-phenylindole; MSC, mesenchymal stem cell.

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MSC Trigger the Appearance of Nestin + Progenitor Cells

The outer layer of the ciliary body pigmented epithelium is home to a quiescent neural progenitor cell (NPC) population that can be detected by expression of the progenitor cell marker nestin [40–46]. As both pigmented and nonpigmented cells were observed in the TM area of MSC- and MSC-CM-treated eyes, we investigated whether these cells could represent NPCs. Staining for nestin + cells was conducted in the chamber angle area: ciliary body, TM, and limited portion of the iris (Fig. 4). In an untreated eye, no nestin + cells were detectable (Fig. 7A). Few nestin + cells were detected in the laser-treated group injected with the lymphocyte control cell population (Fig. 4A). In comparison, a high proportion of nestin + cells were observed in the MSC- and low-oxygen CM-treated groups and were composed of both pigmented and nonpigmented cells (Fig. 4B, 4C). Importantly, all nestin + cells were found to be endogenous to the eye as the selected antibody was rat specific, and nestin expression profiles were identical in eyes treated with MSC and MSC-CM (Fig. 4D).

Nestin Expression and Cellular Proliferation Correlates with Ocular Regeneration

As nestin expression is lost in differentiated neural progenitors and mature cells, we used this marker to investigate progenitor cell evolution during tissue regeneration (Fig. 5; Supporting Information Fig. 6). Intact eyes revealed neither cells positive for nestin nor the proliferation nuclear marker Ki67 (with the exception of the corneal surface, which contains replicating (Ki67 +) cells). A small number of nestin + and Ki67 + cells could be observed in laser-treated eyes injected with lymphocyte controls: 2.5% (nestin) and 1% (Ki67) of total cells in the laser damaged area (Fig. 5A, 5B). Both markers were absent in the intact area of this group. In contrast, an increased number of cells harboring nestin and Ki67 were found in the laser damaged area of MSC-treated rats and peaking on day 4 (Fig. 5C, 5D). Approximately 25% of nestin + cells coexpressed Ki67 (data not shown). Interestingly, the moment of peak expression for nestin and Ki67 corresponded to the time required to lower IOP by 50% in the MSC- and MSC-CM-treated groups. Although MSC were found in the intact area of the eye, this region was composed of a significantly lower number of nestin and Ki67 + cells than the laser damaged area (p < .001).

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Figure 6. Proliferating cells integrate chamber angle structures. Rats were treated with EDU to observe the replicating cells fate. (A–I): Representative images of the intact (A, D, G) and laser damaged (B, C, E, F, H, I) chamber angles are shown at days 8, 16, and 32, both as immunofluorescence and light microscopy images, after laser damage and cell injection. Nuclei were DAPI stained. (J): Compilation of total number of EDU + cells present in the eye at days 8, 16, and 32 after laser damage and low-oxygen conditioned medium injection. Cell numbers (mean ± SEM) were determined by fluorescence microscopy analysis of sequential ocular sections (n = 3 per time point). Scale bar = 100 μm. Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; EDU, 5-ethynyl-2'-deoxyuridine.

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Figure 7. Cellular damage raises ocular tissue sensitivity to MSC secretum. Bar graphs are compilations of nestin and Ki67 + cells after (A) MSC injection or (B) low-oxygen CM injection. Areas observed were both the temporal (checkered red) and nasal (checkered blue) sides of an intact eye or the damaged (red) and intact (blue) areas of a laser-treated eye. Pictures are representative images of each group in both the damaged and the intact area: (C) Intact eye with MSC injection, (D) damaged eye with MSC injection, (E) intact eye with Low-oxygen CM injection, (F) damaged eye with low-oxygen CM injection. Nestin (red), Ki67 (green), and DAPI (blue). Significance: **, p < .01; ***, p < .001. Scale bar = 100 μm. Abbreviations: CM, conditioned media; DAPI, 4',6-diamidino-2-phenylindole; MSC, mesenchymal stem cell.

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In the normoxic CM-treated group, we found only a trend for an increased number of nestin + cells between days 2 and 4 (Fig. 5E, 5F). In addition, no significant differences in nestin and Ki67 expression were observed between the intact and damaged areas in this group. Nestin and Ki67 expressing cells were significantly more abundant in the low-oxygen CM than in the normoxic CM group (p < .01 and p < .05, respectively; Fig. 5G, 5H; Supporting Information Fig. 6). While the injection of CM should lead to an even distribution of paracrine factors in the anterior chamber AH, a higher number of nestin + cells was found in the damaged than the intact area of the low-oxygen CM-treated eyes (p < .05). Collectively, these results indicate that MSC and their secretum have a predominant effect on the activation of NPCs, which primarily occurs in the laser-treated area.

Proliferating Cells Integrate and Survive in Chamber Angle Structures

As both Nestin and Ki67 are transient markers, we investigated the fate of the proliferating cells using EDU staining (Fig. 6). In animals treated with low oxygen CM, the peak of proliferation was observed between days 1 and 3, hence, a group of animals was treated by daily intraperitoneal injections of EDU during that time to stain the proliferating cells. A large number of EDU + cells (6.4 ± 0.4 × 105; Fig. 6B, 6C, 6J) were found in the damaged area of the treated eyes on day 8. Although these cells decreased significantly (p < .001) when observing treated animals on day 32 (5.2 ± 0.4 × 105; Fig. 6H, 6I, 6J), they were still present in large enough numbers to confirm they successfully implanted in the area of damage. No EDU + cells were detectable after 1 month in the intact area (Fig. 6G). This indicates that the Ki67 + cells observed between days 1 and 4 remained, a majority of which were nestin + , in the chamber angle and fully integrated in the ciliary body and TM.

Laser Exposure Raises Local Ocular Tissue Sensitivity to MSC Secretum

In light of the finding that cellular damage promoted local recruitment of progenitor cells, we evaluated whether laser-induced tissue damage had a local or systemic influence on MSC- or MSC-CM-induced progenitor cell recruitment and cellular proliferation. To answer this question, intact and laser-treated eyes were injected with MSC and low-oxygen CM. In an intact eye, no nestin or Ki67 + cells could be detected after MSC injection (Fig. 7A, 7C). In contrast, the injection of the same number of cells in a laser damaged eye led to an increase in nestin and Ki67 + cells, which was predominant in the area of laser damage (Fig. 7A, 7D). In addition, low-oxygen CM increased nestin and Ki67 + cells diffusely in the anterior chamber of both eyes whether they were laser treated or not (Fig. 7B, 7E). Moreover, laser treatment was the only factor causing a differential increase in nestin expression after CM exposure (Fig. 7B, 7F; p < .01). These results indicate that laser exposure enhances cellular sensitivity to MSC resulting in targeted NPC generation and cell proliferation. MSC-CM generated increases in nestin and Ki6 + cells in laser-unexposed eyes or ocular regions, but only laser exposure promoted differential NPC enhancement.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

While embryonic stem cells have been used to repair damaged ocular tissues, to our knowledge this is the first study to report that BM cell subsets harbor regenerative potential, both functional and histological, for the anterior segment of the eye. We identified MSC as prime mediators of this beneficial effect. In addition, our findings demonstrate the unique ability of paracrine factors secreted by low-oxygen cultured MSCs to enhance the function and stimulate the proliferation of ocular progenitor cells. We also report that local stem cell recruitment in the ocular anterior chamber upon exposure to MSC was associated with functional tissue repair. Moreover, laser-induced endogenous progenitor cell stimulation could explain why very short exposure to MSC or their paracrine factors can repair dysfunctional TM. Such an approach opens the door to cellular therapy in patients with chronic conditions, such as glaucoma.

MSC were found to be the crucial BMMC subpopulation inducing a functional regenerative effect and lowering IOP, as ablation of MSC from the injected cells resulted in considerably less efficient tissue repair. Neither injection of saline solution nor lymphocytes could decrease IOP, indicating that IOP drop did not result from nonspecific medium or cell injections. In addition, lymphocytes being particularly prone to induce immune reactivity, the fact that their administration failed to decrease IOP suggests that the decreased pressure did not result from an increased inflammatory response to the graft [47–49]. Interestingly, MSC injected in the anterior ocular chamber persisted only for a few days, far less than the 5 weeks persistence in the retina observed after MSC injection in the vitreous in a similar glaucoma model [50]. However, the duration of MSC infiltration of the TM observed in our study is in line with other studies of intra-arterial cell injection in animal models of stroke where MSC persist only transiently and are no longer found after 7 days [5, 51-53]. This could result from MSC escaping from the injected eye through the TM or from early mortality after migrating to areas of severe cellular stress inhospitable to long-term engraftment [54]. Our findings suggest that such an environment leads to MSC apoptosis and phagocytosis by ocular microglia. Conversely, the observed beneficial effects indicate that long-term engraftment is not required for ocular regeneration and that less than 4 days are sufficient to generate a physiological effect.

The mechanisms by which MSC induce tissue recovery are still widely debated, with cellular differentiation and paracrine effects being the two lead possibilities [8]. Our results clearly favor the latter, as the lack of MSC differentiation in vitro upon exposure to TM cells, their early disappearance from the TM in vivo as well as the rapid decrease of IOP and rise of AH clearance within days of low-oxygen MSC-CM injection, all indicate that MSC likely act through the release of soluble mediators. Moreover, the absence of pressure lowering after injection of unstressed MSC-CM reinforces the need for cellular stress to activate MSC [55–62]. These findings provide clear evidence that MSC can act in a paracrine fashion and exert their protective and regenerative effect without the need for prolonged tissue infiltration.

The ciliary body hosts a cell population known to be quiescent neuronal progenitor cells identifiable by the interfilament protein nestin [40–43]. Nestin is commonly used as a neuronal stem/progenitor cells marker and has been observed in a broad range of regenerating tissues after injury [63–65]. Our data clearly indicate that MSC secretum is responsible for the upregulation of nestin as well as the cellular proliferation marker Ki67 in the TM. The fact that the peak in nestin + cells corresponded to the IOP descent implies a role for NPCs in tissue regeneration or at least identifies these cells as indicators of functional tissue regeneration. Importantly, nestin and Ki67 were expressed only transiently following exposure to MSC and MSC-CM. This is a reassuring finding since uncontrolled cell proliferation could be deleterious, particularly in a closed environment such as the eye. Additionally, cells tagged with EDU during this active proliferation period were present in large numbers in the damaged area 1 month after injury. This indicates that these cells actively take part in tissue remodeling and in functional recovery after regeneration is complete, a phenomenon suggesting that not only tissue protection but also tissue regeneration is occurring. However, the current model does not allow for longer term studies and it will be interesting to challenge not only the long-term regenerative potential of MSC and MSC-CM but also long-term functionality of the regenerated TM.

The use of 180° laser-induced damage in this animal model, with 180° intact area in the same eye, allowed the precision of MSC-induced regeneration to be revealed with striking accuracy. Indeed, the damaged TM area contained a greater proportion of transplanted MSC compared to intact areas of the same eye, indicating that injected cells migrate preferentially to the site of laser treatment. Laser-induced humoral gradients emanating from damaged cells probably contributed to MSC migration, as inflammatory cytokines and chemokines involved in the migration of immune cells toward wound sites are also implicated in MSC mediated chemotaxis [66–68]. In addition, we have observed that nestin and EDU + cells were increased more efficiently in laser-treated areas. Nevertheless, MSC usage resulted in a more localized effect than MSC-CM, thus implying that MSC might need to be in close contact with damaged cells to upregulate their cytokine production and influence cells in their immediate vicinity. Such cytokine upregulation could result from damaged area hypoxia and also from contact with inflammatory factors or other cytokines mediating a form of cellular stress. Moreover, soluble mediators from laser-damaged cells could synergize with MSC secretum to upregulate NPC surface receptors enhancing their response. While the administration of MSC can promote the delivery of numerous therapeutic factors under tightly regulated mechanisms, MSC secretum injection may represent a clinical strategy with broad applicability and low potential for toxicity.

This study has important clinical implications not only for the treatment of acute disorders but also for chronic diseases such as glaucoma. Indeed, one could envision that laser trabeculoplasty, a standard clinical treatment for glaucoma, could be combined with MSC or MSC-CM administration to promote TM regeneration in glaucoma patients. Laser trabeculoplasty would have the advantage of generating local damage and inflammation within the TM architecture necessary for MSC activation, although it does not induce the same degree of inflammation or tissue architectural changes as in our model. It might also remove nonfunctional or dysfunctional TM cells thus creating space for new TM cells. By promoting preferential homing of MSC to the TM, laser therapy would enable them to enhance nestin + stem cell recruitment to replace/repair damaged tissues. However, it remains to be determined whether the lower treatment intensity of laser trabeculoplasty and human MSC would yield the same results in humans as the currently investigated treatment conditions. The fact that neuronal progenitor cells can be found throughout the CNS, and that MSC or their secretum can rapidly recruit NPCs could therefore have implications in the treatment of neurodegenerative disorders outside the eye.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Our data indicates that MSCs targeted to the trabecular meshwork through laser treatment can promote ocular tissue regeneration. Such an effect was mediated by paracrine factors produced by MSCs activated following exposure to low oxygen conditions and was observed in the laser-treated ocular environment. The decrease in IOP was associated with TM cell recovery, enhanced ocular outflow, and progenitor cell recruitment. These results provide a basis for the development of therapeutic strategies aimed at acute and chronic diseases where functional tissue recovery is direly needed.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

We thank Dr. Leonard Levin and Dr. Nicolas Noiseux for their helpful discussions on the manuscript; Dr. Guy Allaire for his expert input on the ocular histology; and Dr. Gilbert Bernier for his expertise on ocular progenitor cells. This study was supported by Glaucoma Research Society of Canada, Thécell Network - Fonds Recherche Quebec - Sante (FRQS), Fonds de Recherche en Ophtalmologie de l'Université de Montréal, the Fonds de recherche Québec - Santé, and Canadian Institutes for Health Research.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
sc-12-0633_sm_SupplFigure1.pdf1873KSupplementary Figure 1. BMMC Characterization. BMMC population was characterized for hematopoietic cells and MSC. A) Using CD45 and CD14 hematopoietic marker, we identified 65% of non hematopoietic cells after 2 passages in vitro. Gated cells were positive for CD73 (B) CD90 (C) and CD105 (D). Selected CD45-CD14 negative cells were plated in adipogenic (E) and osteogenic (F) media, were allowed to differentiate and compared to non treated MSC controls. Representative images of flow cytometry analysis and MSC differentiation. Scale bar = 50 μm
sc-12-0633_sm_SupplFigure2.pdf1876KSupplementary Figure 2. MSC do not transdifferentiate into TM cells in vitro. A) TM cells and MSC before and after 1 week co-culture with TM cells were assessed for the presence of laminin, fibronectin, aquaporin-1 and Pax6 by flow cytometry. Fluorescence intensity was measured on cell tracker positive MSC (dot plot). B) Compilation of mean fluorescence intensity for each antigen in TM cells, MSC alone and MSC co-cultured with TM. (Mean±SEM; n=3 from 3 distinct experiments). Significance: *p<0.05, ***p<0.001.
sc-12-0633_sm_SupplFigure3.pdf1570KSupplementary Figure 3. MSC phagocytosis by ocular microglia. Pictures are representative images of day 2 post laser damage tissue sections that were stained with microglia marker F 4/80 after A) injection of saline solution or B) injection with CFSE+ MSC. Scale bar = 50 μm
sc-12-0633_sm_SupplFigure4.tif1261KSupplementary Figure 4. MSC paracrine factors promote restoration of TM function. Aqueous humor clearance was evaluated by measuring peripheral blood radioactive tracer levels in counts per minutes (CPM) at different time points after injection of radiotracer in normal eyes (black line, n=3), laser treated eyes (blue line, n=3) and low oxygen CM treated eyes (red line, n=3). Results are expressed as mean±SEM of 2 independent experiments. Statistical significance was observed in low O2 CM exposed eyes versus laser only and normal controls: *p<0.05
sc-12-0633_sm_SupplFigure5.pdf1629KSupplementary Figure 5. Decreased IOP allows for better survival of retinal cells Histological sections of the retinal area from a rat eye stained by TUNEL assay. Pictures are representative images of retinas from a A) untreated control B) Laser + normoxic CM treated eye, day 12 C) laser + low oxygen treated eye, day 12. D) Compilation of total number of TUNEL+ cells present in the retina 12 days after laser damage and CM injection. Cell numbers (mean±SEM) were determined by fluorescence microscopy analysis of sequential ocular sections (n=3 per time point). *p<0.05. Scale bar = 100 μm
sc-12-0633_sm_SupplFigure6.pdf1656KSupplementary Figure 6. Nestin expression and cellular proliferation in the damaged and intact areas. Graphs represent compilation of nestin (A-B) and Ki67 (C-D) expressing cells based on their location: Intact area (A-C) and damaged area (B-D). Three animals per group from 2 separate experiments (N=6). Numbers indicate the percentage of positive cells found in the area composed of the ciliary body, the TM and a limited portion of the iris.

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