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Keywords:

  • Adult stem cells;
  • Cell migration;
  • Stem cell transplantation;
  • Experimental models;
  • Tissue regeneration Tissue-specific stem cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

At present, there are severe limitations to the successful migration and integration of stem cells transplanted into the degenerated retina to restore visual function. This study investigated the potential role of chondroitin sulfate proteoglycans (CSPGs) and microglia in the migration of human Müller glia with neural stem cell characteristics following subretinal injection into the Lister hooded (LH) and Royal College of Surgeons (RCS) rat retinae. Neonate LH rat retina showed minimal baseline microglial accumulation (CD68-positive cells) that increased significantly 2 weeks after transplantation (p < .001), particularly in the ganglion cell layer (GCL) and inner plexiform layer. In contrast, nontransplanted 5-week-old RCS rat retina showed considerable baseline microglial accumulation in the outer nuclear layer (ONL) and photoreceptor outer segment debris zone (DZ) that further increased (p < .05) throughout the retina 2 weeks after transplantation. Marked deposition of the N-terminal fragment of CSPGs, as well as neurocan and versican, was observed in the DZ of 5-week-old RCS rat retinae, which contrasted with the limited expression of these proteins in the GCL of the adult and neonate LH rat retinae. Staining for CSPGs and CD68 revealed colocalization of these two molecules in cells infiltrating the ONL and DZ of the degenerating RCS rat retina. Enhanced immune suppression with oral prednisolone and intraperitoneal injections of indomethacin caused a reduction in the number of microglia but did not facilitate Müller stem cell migration. However, injection of cells with chondroitinase ABC combined with enhanced immune suppression caused a dramatic increase in the migration of Müller stem cells into all the retinal cell layers. These observations suggest that both microglia and CSPGs constitute a barrier for stem cell migration following transplantation into experimental models of retinal degeneration and that control of matrix deposition and the innate microglial response to neural retina degeneration may need to be addressed when translating cell-based therapies to treat human retinal disease.

Disclosure of potential conflicts of interest is found at the end of this article.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

Stem cell-based therapies provide a hope for restoration of sight in individuals whose retinal function has been irreversibly damaged by disease. Transplantation of neural stem cells and retinal progenitors has been extensively performed in various experimental models of retinal disease, with several studies yielding a mixed success to date [1, [2], [3], [4], [5], [6]7]. However, before this procedure can be translated into human therapies, many problems need to be solved to promote adequate cell survival, differentiation, and functional integration of grafted cells into the retina. Studies involving retinal transplantation of brain-derived precursor cells into Royal College of Surgeons (RCS) rats have been shown to promote photoreceptor cell survival, but although the transplanted cells migrate to the photoreceptor layer in some instances, they do not express retinal-specific markers [8, [9]10]. It has been suggested that either specific retinal precursors are needed for functional and morphological regeneration, or specific cues, such as retinal injury, are needed for appropriate migration and differentiation of transplanted cells. These views have been confirmed by observations that brain progenitors and ocular stem cells show improved migration, although not optimal integration and differentiation into retinal neurons when transplanted into injured retina [11, 12]. It has also been shown that neonate retina provides an amenable environment for stem cell transplantation [12, 13] and that the ontogenic stage of transplanted retinal precursors determines the ability of these cells to integrate into degenerated retina [14].

Müller glial cells have shown neural regenerative ability in the postnatal retina of zebrafish, chick, and rat [15, [16]17], and a population of Müller glia with neural stem cell characteristics has recently been identified in the adult human eye [18]. These cells have the potential to be used in cell-based therapies to treat retinal disease, but like other stem cells used for experimental transplantation to regenerate retina, when grafted into neonate and degenerated retina, they show limited migration and integration [18].

Retinal degeneration is characterized by formation of glial scarring [19] and severe microglial activation [20, [21]22], which may contribute to the lack of migration, integration, and differentiation of transplanted stem cells. Adult CNS neurons that retain the ability to grow following injury are unable to extend processes beyond the injury-induced glial scar due to the presence of inhibitory proteins such as the chondroitin sulfate proteoglycans (CSPGs) aggrecan, versican, and neurocan [23, 24]. These proteins, which inhibit axon guidance [25], have been identified in the human retina and appear during normal development [26]. Their response to retinal injury has not been widely investigated, but deposition of CSPGs has been shown to inhibit regeneration of the injured rat optic nerve [27]. Degradation of CSPGs using enzymatic digestion by chondroitinase enhances neurite outgrowth and axon regeneration in injured brain [28] and spinal cord [29, [30]31], and it is possible that similar treatments may facilitate functional integration of stem cells transplanted into the retina.

On the basis of the above evidence, we investigated whether CSPG deposition and macrophage/microglia accumulation may prevent the successful migration and integration of Müller glial stem cells when transplanted into the subretinal space of the dystrophic RCS rat, an experimental model of retinal degeneration. Since neonatal retina is known to provide a permissible environment for stem cell transplantation [12, 13], we compared the response of the dystrophic 5-week-old RCS rat retina with that of the neonate Lister hooded rat. We also examined whether CSPG degradation and microglial inhibition could facilitate migration and integration of the grafted cells.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

Animals and Immunosuppression

Dystrophic RCS rats and neonatal Lister hooded rats were used in the study. All rats were maintained according to the Home Office regulations for the care and use of laboratory animals and the U.K. Animals (Scientific Procedures) Act (1986). Dystrophic RCS rats were bred in-house, kept under a 12-hour/12-hour light-dark cycle (light cycle mean illumination, 30 cd/m2), and transplanted at the age of 5–6 weeks (35–40 days), by which time the retinal degeneration is well established. Lister hooded rats were purchased from Harlan (Harlan UK Ltd., Oxon, U.K., http://www.harlaneurope.com/main-uk.htm). Animals were normally immunosuppressed with oral cyclosporine A (210 mg/l of drinking water; Sandimmun; Sandoz, Camberley, U.K., http://www.sandoz.com) and azathioprine (20 mg/l; Sigma-Aldrich, Gillingham, U.K., http://www.sigmaaldrich.com) from 2 days before transplantation until termination of the experiment. When using additional immunosuppression, animals received oral prednisolone (5 mg/l; Sovereign Medical, Bishops Stortford, U.K., http://www.sovereignmedical.org/) in addition to cyclosporine A and azathioprine, together with daily intraperitoneal injections of indomethacin (0.1 mg per 100 g of body weight) for the duration of the experiment. For transplantation into neonatal Lister hooded pups, pregnant dams were immunosuppressed with oral cyclosporine A and azathioprine as described above.

Isolation and Preparation of Müller Cells with Stem Cell Characteristics

Isolation of Müller stem cells from the neural retina of donor human eyes consented for research was performed as previously described [32]. Briefly, neural retina sectioned at least 2 mm away from the ora serrata was incubated in trypsin-EDTA (0.5% trypsin/0.2% EDTA; Invitrogen, Paisley, U.K., http://www.invitrogen.com) for 20 minutes at 37°C. After vigorous trituration, released cells were washed and suspended in Dulbecco's modified Eagle's medium (DMEM) containing GlutaMAX-1 (Invitrogen), 10% fetal calf serum (Invitrogen), and 40 ng/ml epidermal growth factor (EGF; Sigma-Aldrich). Cells were plated onto fibronectin-coated tissue culture dishes and cultured for 2–3 weeks until formation of adherent cell colonies. Colonies were detached and transferred onto new culture dishes, and culture was continued in the above medium without EGF. Upon reaching confluence, cells were examined for their stem cell characteristics as previously described [18]. Cells that underwent more than 50 passages without losing their stem cell characteristics were used for transplantation.

Preparation of Cells for Transplantation

Müller stem cells used for transplantation were transfected with an immunodeficiency virus type 1 (HIV-1)-based, lentiviral vector-expressing, low-toxicity humanized R. reniformis green fluorescent protein from a spleen focus-forming virus promoter, previously described as l-schrgfpw [33]. Confluent cells plated in a 12-well culture dish were infected with l-schrgfpw at a multiplicity of infection of one transducing unit per cell in the presence of polybrene (10 μg/ml; Chemicon, Temecula, CA, http://www.chemicon.com). Using this method, more than 80% of Müller stem cells expressed green fluorescent protein (GFP) 1 week after infection. The transfected cells were grown to confluence in a 25-cm2 flask, and green fluorescent cells were selected by fluorescence-activated cell sorting using a FACSCalibur (BD Biosciences, Oxford, U.K., http://www.bdbiosciences.com). To ensure that the lentiviral vector did not modify the stem cell characteristics of the transfected cells, they were examined for the expression of stem cell markers and sphere formation as previously described [18]. Three days prior to transplantation, lentivirus-GFP-transfected cells were plated onto a 75-cm2 flask and allowed to reach approximately 70% confluence. On the day of the transplant, cells were trypsinized, counted, and resuspended in serum-free medium to a concentration of 2 × 104 cells per microliter.

Subretinal Transplantation

Rats were anesthetized with an intraperitoneal injection of ketamine HCl 7.5 mg/100 g (Ketaset; Fort Dodge Animal Health, Southampton, U.K., http://www.fortdodge.eu/) and medetomidine HCl −5 mg/100 g (Domitor; Pfizer, Sandwich, U.K., http://www.pfizer.com). The pupils were dilated using 1% tropicamide and 2.5% phenylephrine (Chauvin Pharmaceuticals, Surrey, U.K.) before injection of 2 μl of the cell suspension into the subretinal space of adult dystrophic RCS rats (n = 9) using a 30-gauge metal needle attached to a Hamilton syringe, under direct visualization with a Leitz operating microscope. Sham-injected rats received DMEM without cells (n = 6). Neonatal Lister hooded rats (postnatal day 2) were anesthetized with 1/10th the adult dose of ketamine (0.75 mg/100 g) and medetomidine (0.5 mg/100 g) by intraperitoneal injection. The lids were opened surgically, and 1 μl of cell suspension (n = 11) or medium (n = 9) was injected into the subretinal space using a 32-gauge metal needle attached to a 2.5-μl Hamilton syringe. When treating RCS retinae with chondroitinase ABC (n = 8), cells were suspended at a concentration of 4 × 104 cells per microliter. Prior to transplantation, 1 μl of the cell suspension was mixed with 1 μl (0.01U) of chondroitinase ABC (ChABC; Seikagaku, Tokyo, http://www.seikagaku.co.jp/english), and the mixture was injected into the subretinal space as indicated above.

Tissue Processing and Immunohistochemistry

Two weeks post-transplantation, rats were terminally anesthetized with intraperitoneal sodium pentobarbitone and perfused transcardially with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Eyes were excised, postfixed in PFA for 1 hour, cryoprotected with 30% sucrose overnight, and embedded in optimal cutting temperature compound (VWR, Lutterworth, U.K., http://uk.vwr.com/app/Home). Cryostat sections 14 μm thick were placed onto precharged slides, and the slides were air-dried prior to storage at −80°C. Sections with visible transplants (in eyes that underwent cellular transplantation) (untreated RCS, n = 4; neonatal Lister hooded, n = 6; ChABC-treated RCS, n = 5) and sections with visible transplant sites (in sham-operated eyes) (RCS, n = 3; Lister hooded, n = 3) were selected for immunostaining. Sections from two different unoperated eyes in each group were used as controls.

Dried slides were blocked with 5% donkey serum in PBS/0.3% Triton and reacted with primary antibodies diluted in the same blocking solution overnight at room temperature (RT). The primary antibodies used were CSPG (CS56; monoclonal; 1:200; Sigma-Aldrich); neurocan (IF6; monoclonal; 1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/∼dshbwww); versican (12C5; monoclonal; 1:100; Developmental Studies Hybridoma Bank); ED1 (anti-rat-CD68; monoclonal, 1:1,000; Serotec Ltd., Oxford, U.K., http://www.serotec.com), a marker for rat macrophage/microglia; and 2B6 (Seikagaku), an antibody that recognizes an epitope exposed following chondroitinase ABC degradation of chondroitin-4-sulfate. After incubation with primary antibodies overnight at RT, sections were washed in PBS and incubated with the relevant Alexa Fluor secondary antibodies (mouse, rabbit, or goat 488 or 555, raised in donkey; Invitrogen) diluted 1:500 in PBS plus 2% donkey serum for 1.5 hours at RT. After washing, sections were counterstained with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) washed with Tris buffer (0.05 M; pH 7.4), and mounted in Vectashield (Vector Laboratories, Peterborough, U.K., http://www.vectorlabs.com). Sections were also processed in parallel with secondary antibodies alone, which served as negative controls.

Macrophage/microglial infiltration was determined by costaining of retinal sections with a mouse monoclonal antibody to CD68 antigen (ED1; Serotec). Antibody reactivity was determined by visualization with 3,3′-diaminobenzidine (DAB; Sigma-Aldrich) enhanced with aqueous 1% nickel ammonium sulfate and aqueous 1% cobalt chloride by a modification of published methods [34] as follows. Sections were blocked and incubated with the anti-CD68 antibody overnight at RT, using the same blocking solution used with other antibodies in the study (described above), with the addition of 0.5% bovine serum albumin (Sigma-Aldrich). After washing, sections were incubated in biotinylated horse anti-mouse secondary antibody, rat-adsorbed (1:150, Vector Laboratories), followed by streptavidin-horseradish peroxidase (Vector Laboratories). They were then developed with 25 seconds of incubation in DAB solution (0.05% in 0.1 M PBS with 1% nickel ammonium sulfate and 1% cobalt chloride in hydrogen peroxide), and cells expressing CD68 were detected by their characteristic brown-black staining under Nomarski illumination.

To identify other markers in sections stained for CD68, slides were then washed, blocked, and incubated with other primary antibodies (described above) and detected with fluorescence-labeled antibodies. Sections were photographed using a Zeiss LSM 510 confocal microscope, and images were analyzed using the Zeiss LSM Image Browser software (Carl Zeiss, Oberkochen, Germany, http://www.zeiss.com).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

Migration and Integration of Müller Stem Cells Following Subretinal Grafts into Neonate Lister Hooded and RCS Rats

Two weeks after transplantation of Müller stem cells into the subretinal space of neonate (2 days old) Lister hooded (LH) rats, migration of cells into the inner and outer retinal cell layers was often observed. However, relatively few cells migrated, and this migration occurred only into the area adjacent to the transplantation site (Fig. 1A). In contrast, 2 weeks after transplantation of these cells into the subretinal space of 4–5-week-old RCS rats (the age at which the photoreceptor cell layer has been reduced to about half of its normal thickness), cells accumulated at the interface between the outer segment debris zone (DZ) and the retinal pigment epithelium and often gave the appearance of small-cell aggregates or cell debris (Fig. 1B). Cells rarely migrated into the retina. Examination of the whole RCS retina 2 weeks after transplantation showed that cells migrated predominantly along the subretinal space lining the DZ (Fig. 1C). It was of interest that cells that had been grafted in the subretinal space of the dorsotemporal region were often seen along the entire subretinal space, but not within the retina itself (Fig. 1C).

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Figure Figure 1.. Migration of Müller stem cells following subretinal transplantation into the neonate LH and dystrophic RCS rats. (A): Migration of Müller stem cells (green fluorescent protein [GFP]-labeled) into the INL and ONL 2 w after transplantation into a 3-day-old LH rat. (B): Müller stem cells (GFP-labeled) remained in the subretinal space 2 w after transplantation into a 5-w-old RCS rat. (C): Montage of whole retina to show widespread Müller stem cell migration along the subretinal space 5 w after subretinal injection into a 5-w-old RCS rat. Red staining indicates reactivity to an antibody against the neural marker HuD. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Abbreviations: DT, dorso temporal; DZ, debris zone; GCL, ganglion cell layer; INL, inner nuclear layer; LH, Lister hooded; NT, naso temporal; ONL, outer nuclear layer; RCS, Royal College of Surgeons; w, week(s).

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Microglia Response to Retinal Transplantation of Müller Stem Cells

Since microglia are known to migrate and proliferate within the degenerating retina of the RCS rat [20, [21]22], we investigated whether these cells play a role in the inhibition of Müller cell migration following subretinal transplantation into 5-week-old RCS rats. We also compared this response with that of neonatal LH rats. Examination of the presence of microglia in LH neonate retina showed only occasional CD68-positive cells within the ganglion cell layer (GCL) of 2-day-old animals (Fig. 2A). However, 3 weeks after transplantation, an increase in the number of CD68-positive cells was observed in the GCL and inner plexiform layer of LH rats, despite immune suppression with oral cyclosporine and azathioprine (Fig. 2B). Confocal examination of retinal sections from transplanted neonatal LH rats stained for CD68 and GFP showed that although Müller stem cells had migrated into the retina, microglial reactivity was often associated with the grafted cells (Fig. 2C).

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Figure Figure 2.. Distribution of microglia in the LH and RCS rat retina before and after Müller cell transplantation. (A): Neonate LH rat retina (3-day-old animal) showing minimal microglia reactivity as judged by CD68-positive cells (black) in the GCL. (B): Microglia accumulation (black staining) in the GCL and IPL of an LH rat retina 2 wks after neonatal transplantation. (C): Müller stem cell (green fluorescent protein-labeled) migration into the GCL 2 wks after transplantation into the neonate LH rat retina (left). Middle panel shows Nomarski illumination of the same section, demonstrating accumulation of CD68-positive microglia (black) in the GCL. Right panel illustrates Nomarski illumination of the same section, showing association of microglia with transplanted Müller stem cells. (D): Nomarski illumination of a nontransplanted RCS rat retina at 5 wks of age showing severe infiltration of microglia in the outer segment DZ and the ONL. (E): Nomarski illumination of RCS rat retina 2 wks after transplantation in a 5-wk-old animal. Microglia (black) can be observed thorough all retinal cell layers. (F): Histogram shows a significantly higher number of microglia in 5-wk-old RCS rat retina compared with the neonatal LH retina (*, p = .0055). Results are the mean ± SE of the mean of three different experiments. (G): Following Müller stem cell transplantation, there was a significant increase in microglial accumulation in both the LH (*, p < .001, transplanted vs. nontransplanted retina) and the RCS (**, p < .05, transplanted vs. nontransplanted) rat retinae. Results are the mean ± SE of the mean of three different experiments. Abbreviations: DZ, debris zone; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; LH, Lister hooded; NR, neural retina; ONL, outer nuclear layer; RCS, Royal College of Surgeons; wks, weeks.

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Unlike the mild microglia activation observed in the transplanted neonatal LH retina, a marked accumulation of CD68-positive cells was observed in the retina of 5-week-old dystrophic RCS rats. Numerous microglia were observed in the outer segment DZ and in the outer nuclear layer (ONL) (Fig. 2D). Two weeks after Müller stem cell injection into the subretinal space, adult RCS rat retina showed a more severe and widespread migration of microglia (Fig. 2E), despite immune suppression with oral cyclosporine and azathioprine. Quantitative comparison of the infiltrating microglia in the LH and RCS rat prior to and following transplantation showed that the degenerating RCS rat retina harbored significantly higher numbers of microglia than the normal LH rat retina (p < .01) (Fig. 2F) and that Müller stem cell transplantation caused a marked increase in the number of microglia in both the LH (p < .001) and RCS (p < .05) rat retinae (Fig. 2G).

Confocal microscopy of transplanted retinal sections costained for CD68 and GFP showed that Müller stem cells that had not migrated into the retina and that lined the outer segment DZ were closely associated to microglia, as judged by the colocalization of CD68 and GFP (Fig. 3A). Moreover, aggregates of grafted Müller stem cells, frequently observed in the subretinal space of the transplanted RCS rat, were always surrounded by microglia (Fig. 3B). To promote cell migration by diminishing microglial activation, animals were given oral prednisolone and daily intraperitoneal injections of indomethacin, in addition to oral cyclosporine and azathioprine, for the duration of the experiment. As observed in Figure 3C, although microglial numbers appeared comparatively reduced with additional prednisolone and indomethacin treatment, transplanted cells continued to be associated with microglia and remained in the DZ, unable to migrate into the retina. Although better migration of transplanted cells into the retina of the neonate LH rat was generally observed, on occasions when trauma to the retina occurred as a result of the transplantation procedure, severe microglia accumulation at the site of the injection also prevented Müller stem cells from migrating into the retina of these animals (Fig. 3D).

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Figure Figure 3.. Microglia associates with aggregates of Müller stem cells. (A): Left panel shows accumulation of Müller stem cells (green fluorescent protein-labeled) in the subretinal space 2 weeks after transplantation in a 5-week-old Royal College of Surgeons (RCS) rat. Middle panel shows Nomarski illumination to identify CD68-positive cells (black) in the same retinal section. Right panel shows Nomarski illumination to identify colocalization of transplanted cells with microglia. (B): Left panel shows a large cluster of Müller stem cells in the subretinal space 2 weeks after transplantation in a 5-week-old RCS rat. Middle panel shows Nomarski illumination of the same section indicating localization of CD68-positive microglia around the transplanted cells (black). Right panel shows microglia (black) surrounding the transplanted cells and resembling a granuloma-type structure. (C): Retinal section of an RCS rat treated with oral prednisolone and intraperitoneal indomethacin in addition to cyclosporine A and azathioprine. Left panel shows limited migration of grafted cells into the DZ and reduced infiltration of microglia in the same region as that observed under Nomarski illumination in the right panel. (D): Retinal section of a Lister hooded rat 2 weeks after neonatal transplantation in which retinal damage occurred. Animal was not treated with additional microglial suppression. Left panel shows lack of migration and accumulation of Müller stem cells in the subretinal space of the damaged retina. Right panel shows microglia accumulation (black) around the transplant, as observed under Nomarski illumination. Abbreviations: DZ, debris zone; INL, inner nuclear layer; ONL, outer nuclear layer.

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Expression of CSPGs in the Normal and Degenerated Rat Retina and Association of These Proteins with Microglia

Immunostaining of the unoperated 5-week-old RCS rat retina for the CSPG N-terminal region (common to all CSPGs) revealed marked expression of this molecule in the outer segment DZ (Fig. 4A). Strong staining for neurocan and versican was also observed in the same region in unoperated (Fig. 4A) and sham-operated animals (not shown). This contrasted with the expression of these proteins observed in the neonate LH rat, where staining for these molecules was detected only in the ganglion cell region (Fig. 4B).

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Figure Figure 4.. Expression of CSPGs in the retina of nontransplanted Lister hooded (LH) and dystrophic Royal College of Surgeons (RCS) rats and CSPG colocalization with microglia. (A): Confocal images showing accumulation of the N-terminal region of CSPGs, neurocan, and versican (green staining) in the DZ of retinal sections from a nontransplanted 5-week-old RCS rat. (B): Retinal sections of nontransplanted neonate LH rat retina showing expression of CSPGs in the inner plexiform layer and developing GCL. (C): Confocal retinal images of nontransplanted 5-week-old RCS rats showing accumulation of the N-term of CSPGs, neurocan, and versican (green fluorescence) on the left, and colocalization of these proteins with CD68-positive microglia (black) as observed under Nomarski illumination on the right. Cells surrounded by squares are magnified in the insets to show details of microglial colocalization with CSPGs. Abbreviations: CSPG, chondroitin sulfate proteoglycan; DZ, debris zone; GCL, ganglion cell layer; INL, inner nuclear layer; NR, neural retina; N-term, N terminus; ONL, outer nuclear layer.

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Costaining for CSPGs and CD68 in retinal sections from 5-week-old RCS rats showed that microglia were localized mainly at the sites where accumulation of CSPGs could be observed (Fig. 4C). Furthermore, microglia observed outside the DZ and infiltrating the ONL costained for the N terminus of CSPGs, as well as neurocan and versican, suggesting that microglia may constitute a source of CSPGs in the degenerating RCS rat retina (Fig. 4C).

Inhibition of Müller Stem Cell Migration by CSPGs

To elucidate the role of CSPGs on the inhibition of Müller stem cell migration and integration 3 weeks after transplantation into the subretinal space of 5-week-old RCS rats, we examined retinal sections of grafted animals for the expression of CSPGs and CD68. Confocal microscopic analysis of grafted RCS rat retinae showed that Müller stem cells that failed to migrate into the retina were often surrounded by the N terminus of CSPGs, neurocan, and versican (Fig. 5A). CSPGs often formed a pericellular cuffing with severe microglial activation. Colocalization of microglia with all the CSPGs investigated was observed in all specimens examined (Fig. 5A). This spatial correlation of ECM proteins and CD68 expression around the transplanted cells suggests that in addition to CSPGs accumulating in the degenerating retina, CSPGs released by activated microglia are also likely to contribute to the inhibition of cell migration and integration. Further confirmation that absence of CSPGs facilitates migration of the transplanted cells was shown by observations that areas of cell migration did not stain for CSPGs in retina treated with ChABC at the time of transplantation (Fig. 5B).

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Figure Figure 5.. Inhibition of Müller stem cell migration by chondroitin sulfate proteoglycans. (A): Confocal images of retinal sections from 7-week-old Royal College of Surgeons (RCS) rats 2 weeks after subretinal transplantation of Müller stem cells. Sections in the left column show the transplanted cells (GFP-labeled) surrounded by N-CSPG, neurocan, and versican (red). The middle column shows the same sections under Nomarski illumination to illustrate the accumulation of CD68-reactive microglia (black). The column on the right shows the merged images under Nomarski illumination illustrating colocalization of CD68-positive cells and CSPGs (red) surrounding the transplanted cells (GFP-labeled). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). (B): Confocal image of a 7-week-old RCS rat retina 2 weeks after transplantation in the presence of chondroitinase ABC. Staining for the N terminus of CSPGs (red) showed that cells that had migrated (GFP) were not surrounded by these extracellular matrix proteins. Migratory cells are shown within white squares, and their magnification is indicated by red arrows. Abbreviations: CSPG, chondroitin sulfate proteoglycan; GFP, green fluorescent protein; N-CSPG, N-terminal chondroitin sulfate proteoglycan.

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Effect of Combined CSPG Digestion and Microglial Suppression on the Migration of Grafted Müller Stem Cells

To further characterize the contribution of CSPG to inhibition of graft cell migration and integration, we transplanted 5-week-old RCS rats with Müller stem cells together with ChABC to promote matrix degradation and promote cell migration. We also used enhanced microglial suppression on these animals, combining oral cyclosporine A, azathioprine, and prednisolone with daily intraperitoneal injections of indomethacin for the duration of the experiment. Two weeks post-transplantation, we saw a dramatic improvement in the migration of grafted cells through the entire thickness of the retina in eyes treated with ChABC (Fig. 6A). Many of the migrating cells, interestingly, bore characteristic neuronal morphology (Fig. 6A). We quantified this change in migration by counting the total number of grafted cells detected in the retina and classifying them on the basis of their localization to the subretinal space or to the inner retinal layers beyond the ONL. Almost 80% of the cells were found to have migrated into the inner retinal layers when transplanted in conjunction with ChABC. This was in contrast to the control animals, where almost all of the cells remained in the subretinal space (t test; n = 4; p = .0011).

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Figure Figure 6.. Digestion of CSPGs and enhanced microglial suppression facilitates migration of grafted Müller stem cells. (A): Micrograph on the left shows a confocal image of a retinal section from a 7-week-old RCS rat 2 weeks after Müller stem cell transplantation in the presence of ChABC and enhanced immune suppression for the duration of the experiment. Transplanted cells (green fluorescent protein [GFP]-labeled) were observed throughout the whole width of the retina. Middle panel shows the same retinal section under Nomarski illumination to illustrate retinal infiltration by CD68-positive cells (black; arrows). Right panel shows the same section under Nomarski illumination to illustrate the colocalization of CD68-positive microglia with the migrated cells (GFP-labeled; arrows). (B): Confocal images from retinal sections of a 7-week-old RCS rat 2 weeks after injection of ChABC. Sections stained for CSPGs showed a marked reduction in the expression of the N-terminal region of CSPGs, neurocan, and versican (green fluorescence). (C): Left panel shows a retinal section of a 5-week-old nontransplanted RCS rat staining for the stub epitope. Spontaneous degradation of CSPGs is shown by the red staining. Right panel shows a retinal section of a 7-week-old rat 2 weeks after subretinal injection of ChABC. Red staining indicates the exposure of the stub epitope upon degradation of CSPGs by this matrix-degrading enzyme. (D): Histogram shows that subretinal injection of Müller stem cells into RCS rats induced a marked increase in the number of infiltrating microglia (*, p < .05 vs. nontransplanted animals) and that enhanced immune suppression induced a significant reduction in the number of infiltrating retinal microglia (**, p < .001 vs. transplanted animals). (E): Histogram depicting increased migration of Müller stem cells into the inner retinal layers when transplanted with ChABC. No cells were detected beyond the ONL in the control animals (−ChABC), whereas nearly 80% of cells migrated beyond the INL into the inner retinal layers with ChABC (*, p = .001). Results are the mean ± SEM of four different experiments. Abbreviations: ChABC, chondroitinase ABC; DZ, debris zone; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RCS, Royal College of Surgeons; Ab, antibody.

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Reduction in CSPG expression in eyes treated with ChABC was confirmed by a decreased staining for CSPG N terminus, neurocan, and versican in the outer segment DZ (Fig. 6B) compared with untreated retinae (Fig. 4A). In addition, to confirm that reduction in CSPG expression was indeed a result of ChABC digestion, we stained untreated and ChABC-treated retinal sections with the chondroitin sulfate (CS) stub IB5 antibody, which detects CS stub epitopes exposed on CSPG molecules by ChABC enzymatic digestion. As seen in Figure 6C (+ChABC), the ChABC-treated retinae showed widespread staining for the stub antibody throughout the whole retinal thickness, indicating that CSPGs were reduced as a result of ChABC digestion. Interestingly, untreated retinae also showed a localized staining for the CS stub epitopes in the outer segment DZ (Fig. 6C, −ChABC), suggesting that some degree of CSPG digestion also occurs during photoreceptor degeneration. Despite the ChABC enzymatic activity, the retinal architecture of treated animals appeared to be well preserved (Fig. 6A).

With the use of enhanced microglial suppression in the ChABC-treated animals, in addition to improved migration of grafted cells, we also saw a decrease in the amount of microglial accumulation (Fig. 6A). Quantitative analysis showed that the number of microglia within the retinae of animals treated with ChABC and enhanced immune suppression was significantly lower than in animals transplanted without additional immune suppression (Bonferroni p < .001) (Fig. 6D). In addition, the number of Müller stem cells present within the retinae of animals treated with ChABC combined with enhanced microglial suppression was significantly higher than in the animals treated without ChABC (p < .001) (Fig. 6E). Despite the reduction in microglial infiltration, many cells that had migrated and acquired neural morphology were colocalized (although less frequently) with CD68-positive cells (Fig. 6A).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

Successful migration and integration of stem cells to restore tissue function may depend on the appropriate interaction of the host environment with the transplanted cells. Various studies have shown that when stem cells are transplanted to regenerate retina, migration and integration of grafted cells is better in an immature retina or in a retina subject to acute injury [11, 27]. However, little consideration has been given to the migration and integration of stem cells in long-standing retinal degeneration and inflammation. It is important to understand that tissue that needs repair has often undergone a series of inflammatory and degenerative changes, which may limit the functional integration of grafted stem cells. The present observations confirm previous reports that the neonatal retina provides a permissive environment in which stem cells can migrate and integrate. However, our results show that although grafted Müller stem cells migrated into the neonate LH retina, they were often found in association with cells expressing CD68, a marker of macrophage/microglia. Although recent evidence suggests that microglia not only promote neurotoxicity but also have a neuroprotective role and enhance nerve repair [35], it might be possible to speculate that they closely associate with the transplanted cells to promote their migration and survival. However, the present observations that extensive microglial activation was frequently associated with poor migration and survival of Müller stem cells strongly suggest that microglia may have been responsible for inhibiting cell graft migration and integration.

In accordance with previous studies, considerable microglial activation was seen in the degenerating retina of the dystrophic RCS rat prior to transplantation [20, [21]22]. It is therefore likely that pre-existing activated microglia may constitute a first line of response to retinal stem cell transplantation, as they may be ready to exert their cytotoxic and phagocytic functions [35]. Stem cell clusters were often seen in the subretinal space of the RCS rat, densely surrounded by microglia and resembling granuloma-like structures. These clusters were not observed in the neonate LH rat retina, indicating that in the absence of microglia, the grafted cells were able to migrate into the retina. It is possible that the xenograft nature of the transplant (human cells to rat retina) further enhanced the microglia response, but allogeneic transplantation of Müller stem cells in the RCS rat retina resulted in similar microglia reactivity (data not shown).

CSPGs are produced during glial scarring in the central nervous system [24], and their role as inhibitory axon guidance molecules [25, 36] is well documented. We observed, in accordance with other studies, that the retina of the dystrophic RCS rat shows heavy accumulation of the N terminus of chondroitin sulfate proteoglycans [37], neurocan [38], and versican. Our results further demonstrated that microglia, which are known to produce CSPGs in vitro [39] and in vivo upon spinal cord injury [40, 41], also express the N-terminal fraction of CSPGs, as well as neurocan and versican in the degenerating RCS rat retina. Significantly, costaining of retinal sections from transplanted RCS rat retinae for CSPGs and CD68 expression showed that microglia surrounding the grafted cells stained for CSPGs. These observations suggest that one of the mechanisms by which microglia might be inhibiting stem cell migration and integration into the damaged retina is by releasing CSPGs.

Since stem cell migration from the subretinal space into the damaged or injured retina is crucial to retinal repair, we aimed to facilitate cell migration by inhibiting the microglial response in RCS rats using intraperitoneal indomethacin and a combination of oral prednisolone, cyclosporine, and azathioprine [18]. Despite a reduction in microglial accumulation in the vicinity of the transplants as a result of this treatment, residual microglial reactivity was still observed in association with the grafted cells. Microglia remained in the DZ, where CSPG accumulation was observed in the degenerating retina, indicating that microglial suppression alone is not sufficient to promote migration of the grafted cells.

Degradation of CSPGs by chondroitinase has been shown to promote migration of transplanted cells and regenerating axons through glial scars, particularly in the spinal cord [29, [30]31], and in the retina it has been used to improve lentiviral vector-mediated transduction of photoreceptors [42]. Extensive work on the role of CSPG in neuronal plasticity has also revealed that the deposition of this extracellular matrix protein is inhibitory to the formation of new neuronal synaptic connections and is a mechanism by which the mature mammalian nervous system protects itself from the formation of aberrant neuronal synapses when injured [43]. In addition, ChABC has been shown to restore synaptic plasticity by breaking down the CSPG-rich perineural nets in the visual cortex of mature rats [44, 45], and more recent research showed that this enzyme causes improved synapse formation of transplanted photoreceptor precursors with host neurones [46]. On this basis, the role of ChABC might extend beyond its effect on cell migration by facilitating neurite extension of the transplanted cells, as suggested by the observation that Müller stem cells also adopted a neuronal morphology when grafted in the presence of this enzyme.

The present observations that ChABC, in conjunction with oral azathioprine/cyclosporine and intraperitoneal indomethacin, was able to dramatically improve migration of transplanted Müller stem cells into the retina strongly indicate that abnormal deposition of extracellular matrix and activation of the innate inflammatory responses constitute major barriers to retinal stem cell transplantation. Development and refinement of approaches to simultaneously modify the extracellular matrix and suppress microglia reactivity may therefore help to overcome the present limitations to achieve integration of stem cells into degenerated retina. However, further investigation is needed before effective cell-based therapies can be implemented for the treatment of human retinal disease.

Disclosure of Potential Conflicts of Interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

The authors indicate no potential conflicts of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

This work was supported by the Medical Research Council of the United Kingdom (Grant 67386), the Helen Hamlyn Trust (in memory of Paul Hamlyn), and the Henry Smith Charity. S.S. was supported by the Inlaks Foundation, India, and the Henry Smith Trust. P.T.K. was partially funded by the Department of Health's National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital. We thank Dr. A. Vugler for providing the technique for horseradish peroxidase detection of CD68.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References