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a Wallenberg Retina Center, Department of Ophthalmology, Lund University Hospital;
b Wallenberg Neuroscience Center, Department of Physiological Sciences, Lund University, Lund, Sweden
Key Words. Brain-derived precursor cells • RN33B • Subretinal transplantation • Cell markers • Whole-mounted retina • Migration
Anita Blixt Wojciechowski, M.D., Department of Ophthalmology, Lund University Hospital, S-221 84 Lund, Sweden. Telephone: 46-46-2220769; Fax: 46-46-2220774; e-mail: a.blixt{at}bredband.net or anita.blixt_wojciechowski{at}oft.lu.se
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ABSTRACT |
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INTRODUCTION |
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During the second half of the 1980s, the field of experimental retinal tissue transplantation expanded greatly [1, 2]. The method aims at reconstructing the degenerating retina by subretinal grafting of healthy pieces of retinal tissue, which, upon integration with the host retina, will form a functional unit. A few clinical trials using such retinal transplants have been performed, but so far only limited improvement of visual function has been noted [3–8].
Instead of using retinal tissue transplants, in vitro expanded multipotent neural precursor cells could serve as donor cells. Currently, protocols are well established for genetic and epigenetic expansion of rodent and human embryonic and adult neural precursor cells. In several experimental intracerebral transplantation studies, such cells have shown a great capacity to survive, migrate, and differentiate into region-specific neurons with appropriate axonal projections as well as to form astrocytes and oligodendrocytes [9–11].
Retinal transplantation of precursor cells has been performed in order to investigate the possibilities to replace diseased retinal cells and/or to rescue diseased retinal cells in situ by cell-mediated delivery of survival factors. Indeed, transplantation of various neural precursor cells into the retina has been carried out with good cell survival in the normal neonatal and adult host retina [12–18], degenerating retina [19–23], and injured retina [24–26].
To some extent, migration of grafted precursor cells, such as adult rat hippocampal progenitor cells and neonatal rat retinal precursor cells, into the host retina has been observed [12, 13, 19]. We recently described extensive migration in a long-term transplantation experiment using the cell line RN33B, revealing in retinal sections the distribution of RN33B cells from one eccentricity of the adult host retina to the other [17]. Although good retinal graft survival has been estimated by several investigators, no exact number of transplanted cells has been presented, probably due to technical limitations. However, attempts have been made to quantify the survival of grafted cells on cryoprotected sections of the host [12, 13, 19]. In the present study, the brain-derived precursor cell line RN33B was further studied. The cells are genetically immortalized with the temperature-sensitive simian virus (SV40) large T antigen. In order to detect the cells post-grafting, the RN33B cell line is genetically labeled with the reporter genes LacZ and the green fluorescent protein (GFP) [27–29].
The purpose of the present study was primarily to estimate survival and assess migration, but also to study integration and differentiation of grafted RN33B cells in whole-mounted retinas.
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MATERIALS AND METHODS |
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The cell line previously had been retrovirally transduced with the reporter gene LacZ [27] and was additionally transduced with the GFP gene using a lentiviral vector [28, 29].
Transplantation
On the day of transplantation, the cell culture medium was replaced with Hank’s balanced salt solution (HBSS; GIBCO, Life Technologies) containing 0.1% trypsin. When the cells were detached, serum-containing medium was added. The cells were then centrifuged, resuspended, and counted in a hemocytometer and finally prepared into a suspension of 100,000 cells/µl in HBSS. At three different transplantation time points, adult SD rats (n = 59; BK Universal; Uppsala, Sweden) were grafted (Table 1
). The left eyes of the animals were injected according to a previously described method [16, 31] with 2 µl of RN33B cells. A 23-gauge needle (Becton Dickinson; Franklin Lakes, NJ; http://www.bd.com) with the tip bent in a 105-degree angle was used to gently open the sclera and the choroid on the superotemporal side of the left eye, between the ora serrata and the equator. Thereafter, a paracentesis was made at the limbus at the nine o’clock position to lower intraocular pressure. The cell suspension was slowly injected using a GELoader Tip (1–10 µl, Eppendorf; Hamburg, Germany; http://www.eppendorf.com) through the sclerotomy. The injection was completed with a 0.1–0.3-µl air bubble to prevent reflux of the cells. A localized retinal detachment could be seen during the injection through the almost transparent sclera and afterwards through the pupil, which indicated that the cell suspension was delivered subretinally. Additionally, seven control rats were subretinally injected with the HBSS medium only. At surgery, the animals were anesthetized using equithesin (pentobarbital 9.72 mg/ml, chloral hydrate 42.5 mg/ml, magnesium sulphate 86.25 mM, 10% vol/vol ethanol, 40% vol/vol propylene glycol, 3 ml/kg body weight i.p.). All animal-related work was carried out according to local ethical guidelines and approved animal care protocols.
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The graft recipients were immunosuppressed with daily injections of cyclosporin A (10 mg/kg i.p.; Sandimmun Neoral, Novartis; Täby, Sweden; http://www.novartis.com). The immunosuppressed rats were also given antibiotics (2 ml/l in drinking water; Borgal, Hoechst Roussel Vet; Münich, Germany; http://www.archive.hoechst.com).
Whole-Mount Preparation
Three and 8 weeks post-transplantation, 31 rats (28 RN33B transplanted rats and 3 HBSS controls) and 35 rats (31 RN33B transplanted and 4 HBSS controls), respectively, were sacrificed with an overdose of CO2 (Table 1
). The eyes were enucleated and placed in 4% paraformaldehyde (PF) for 10–20 minutes. The anterior segment and the lens were then removed; the sensory retina was subsequently dissected, placed on a filter paper (type HA, Millipore; Molsheim, Germany; http://www.millipore.com), and post-fixed in 4% PF for 2–3 hours. Retinas were processed in rising concentrations of sucrose containing Sörensen’s phosphate buffer (pH 7.2) and mounted onto glass slides.
Immunohistochemistry
The whole-mounted retinas were exposed either to a polyclonal rabbit primary anti-serum against ß-galactosidase (ß-Gal; 1:500, 5Prime-3Prime, Inc.; Boulder, CO) in a moist chamber (16–18 hours at 4°C) or to a polyclonal chicken primary anti-serum against GFP (1:5,000, Chemicon; Temecula, CA; http://www.chemicon.com) in a moist chamber for 48 hours at 4°C, followed by rinsing in 0.1 M phosphate-buffered saline with 0.25% Triton-X-100 (Pharmacia Biotech; Uppsala, Sweden; code No 17-1315-01). The retinas were incubated with anti-rabbit Texas Red-conjugated or anti-rabbit fluorescein isothiocyanate (FITC)-conjugated, and/or anti-chicken FITC-conjugated secondary antibodies (1:200, Jackson Immunoresearch; West Grove, PA; http://www.jacksonimmuno.com) for 1–2 hours in room temperature in the dark.
Ten of the retinas transplanted with RN33B cells (two from the 3-week group and eight from the 8-week group), pretreated in 2 M HCl for 10 minutes at 37°C, were used for SV40 large T antigen immunohistochemistry (1:100, Santa Cruz, CA). In addition, two retinas injected with the HBSS medium alone were subjected to the same treatment.
The specimens were examined in an epifluorescence microscope. Distribution patterns of ß-Gal- and GFP-expressing cells were examined by superimposing separate digital images of the fluorochromes. As similar results were obtained with confocal microscope analysis (Bio-Rad 1024 UV laser scanning), we found it more convenient to use the superimposed images.
Cell Counting
Retinas with the most intensive endogenous GFP fluorescence and the widest distribution area of grafted cells were chosen for cell counting (3 weeks, n = 6 and 8 weeks, n = 6). A high fluorescence intensity was required since the counting method exposes the retinas to intense light, which causes fading of the fluorescence. Each of the 12 whole-mounted slides was counted using a semiautomatized stereological cell counting system (CAST-Grid software version 1:10; Olympus; Albertslund, Denmark; http://www.olympus.com) composed of an Olympus BX50 microscope connected to an X-Y-Z-step motor stage.
The retinal area covered with transplanted GFP-expressing cells was delineated in the whole-mounted retina, and a counting frame was randomly placed in the delineated area to mark the first sampling point before being systematically and randomly moved through the entire area. The procedure was then repeated using the Texas Red filter to count the ß-Gal-expressing cells within the delineated area. Data were then extrapolated according to a stereological algorithm: N = SQ x F1 x F2 x F3 (N = total number of marker-expressing cells; SQ = sum of cells counted; F1 = fraction of sections used; F2 = fraction of tissue depth used to collect data; F3 = fraction of tissue area used to collect data) [32]. Delineation of the entire retina was also performed in order to achieve the total retinal area (Table 2).
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RESULTS |
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). Therefore, the 3-week and the 8-week groups with the same survival time were pooled.
Survival of the Transplanted RN33B Cells
Post-transplantation ß-Gal- and GFP-expressing cells were found in 68% (19/28) and 35% (11/31) of the grafted animals at 3 and 8 weeks, respectively (Table 1). A large fraction of the transplanted cells coexpressed ß-Gal and GFP (Fig. 2A and 2B). Images superimposed on each other showed ß-Gal/GFP double-labeling of transplanted cells, as well as subpopulations expressing either ß-Gal or GFP (Fig. 2C and 2D). Smears of the RN33B cells taken prior to transplantation revealed that close to 100% of the cells expressed endogenous GFP (Fig. 1). In control animals, untreated or injected with HBSS medium alone, neither ß-Gal nor GFP immunoreactivity were found.
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). At 8 weeks, the ß-Gal expression was too weak for an appropriate analysis, and quantification of ß-Gal-expressing cells was only possible in one retina out of six, which displayed 31,985 ß-Gal-expressing cells. The average number of GFP-expressing cells in retinas with the most intense fluorescence was estimated as 22,359 (± 10,058) at 8 weeks (n = 6), which indicates that from 3 weeks to 8 weeks post-grafting, the average number of transplanted GFP-expressing cells had increased about 16 times.
Four of the grafted retinas (3 weeks, n = 2 and 8 weeks, n = 2) were exposed to anti-ß-Gal and FITC-conjugated secondary antibodies in order to reveal the total population of grafted cells expressing one or both transgenes. The retinas were then further immunoprocessed using anti-SV40 and Texas Red secondary antibody and analyzed for coexpression of ß-Gal/GFP and SV40. Cells were observed immunoreactive to ß-Gal/GFP and SV40, as well as cells only immunoreactive to SV40 (Fig. 2E). Six of the 20 retinas with no detectable ß-Gal or GFP immunoreactivity from the 8-week group were exposed to anti-SV40; no SV40 immunoreactivity was found. Control retinas (n = 2) injected with the HBSS medium alone and untreated retinas did not express SV40 antigen.
Distribution and Migration of the Transplanted RN33B Cells
With the semiautomatized cell counting system, the distribution area of GFP-positive grafted cells could be delineated. The population of GFP-positive cells was easier to distinguish, thus the distribution area of grafted cells was delineated according to these cells. Since the distribution area of the ß-Gal-expressing cells overlapped the area of GFP-expressing cells at both survival times, this was not considered to introduce a significant bias. The total adult retinal area was estimated to an average of 60.17 (± 7.84) mm2 using values obtained from the nine retinas measured (Table 2). At 3 weeks, RN33B cells were found on a restricted retinal area of, at most, 12.57 mm2 (mean, 5.55 ± 3.75) and on an area of up to 40.8 mm2 (mean, 28.96 ± 11.85) 8 weeks following grafting (Table 2). This correlates to a migratory capacity of the RN33B cells to be distributed throughout a maximum of 21% and 68% of the entire host retina at 3 and 8 weeks, respectively.
Although the RN33B cells migrated over long distances, a large fraction of the grafted cells was found at or close to the implantation site at both 3 and 8 weeks post-grafting. Aggregates of marker-expressing cells were found in the surrounding retina and single scattered cells were seen far from the implantation site (Fig. 3).
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). These cells had morphologies of migrating cells, with elongated cell bodies and an apical process in the direction of the migration [33]. A leading process was demonstrated on both single migrating cells as well as on the cell tubules. The chains of transplanted cells were often arranged radial to the optic nerve head but not along the great retinal vessels (Fig. 5).
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Both at 3 weeks and 8 weeks post-transplantation, the RN33B cells displayed highly variable morphologies and sizes (Figs. 2C and 2D, 6A and 6B). Differentiated RN33B cells did not differ in morphology between the 3- and 8-week retinas (Fig. 2C and 2D). The grafted cells adapted both neuronal and glial-like morphologies. The cellular profiles included bipolar-like cells with small round or long slender somas and multipolar cells with different sizes of the cell somas. Most of the cells had varying numbers of processes, many of them being branched with multiple neuronal-like spines (Figs. 2B, 6C, and 6D). The cell processes were often in close contact with each other, indicating a cell-to-cell contact (Figs. 2C, 2D, and 6C).
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DISCUSSION |
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Survival of the Transplanted RN33B Cells
In the present study, the survival and number of grafted RN33B cells was based on the expression of the reporter genes for GFP and ß-Gal. The GFP marker has proven to possess a long-term and stable expression after transplantation, to be specific for the transplanted cells, and to not be transported to host cells [28, 29]. Here, expression of both the GFP and ß-Gal transgenes was observed up to 8 weeks post-transplantation on whole-mounted retinas. Significant numbers of GFP- and ß-Gal-positive cells were demonstrated on 68% of the transplanted eyes at 3 weeks and 35% at 8 weeks post-transplantation. The mechanism(s) behind the major drop in survival following transplantation, which is as low as zero survival in 32% (at 3 weeks) and 65% (at 8 weeks) of the eyes, is unclear. However, the decline in survival rate at longer survival times might be due to slow rejection of the RN33B cells.
Our recent studies demonstrate that RN33B cells continue to proliferate after transplantation, judging from the expression of the proliferation markers proliferating cell nuclear antigen and SV40 large T antigen [17, 22, 29]. In the brain, only immature cell profiles express SV40 [29]. It might therefore be suggested that RN33B cells expressing only SV40, and not ß-Gal or GFP, are newly formed precursors that have downregulated the reporter genes. However, it cannot be excluded that the persistence of SV40 expression in vivo reflects a loss of the temperature sensitivity of the RN33B cells. The larger number of GFP-positive cells (average, 22,359) estimated by stereological counting at 8 weeks compared with 3 weeks (average, 1,351), further confirms the mitotic activity of RN33B cells after implantation into the adult retina.
The variation of expression of reporter genes (as used here) depends on, for example, vector construct, donor cell type used, age of recipient, and survival time [28, 36]. A study by Lundberg et al. [37], with ß-Gal-expressing RN33B cells labeled with the nuclear marker [3H]thymidine prior to transplantation to adult brain, demonstrated fewer cells that expressed ß-Gal compared with [3H]thymidine post-grafting. Taken together, in order for the most accurate estimation of the number of surviving grafts and cell numbers, a marker(s) independent of the reporter gene is required.
Several intracerebral experimental transplantation studies report overall high cell survival rates after grafting of different in vitro expanded neural precursor types to various regions and ages of the central nervous system [9–11]. Results from retinal transplantation of similar cell types are sparse. Young and coworkers [19] report that at least 50% of the GFP-transduced adult rat hippocampal progenitor cells (AHPCs) transplanted intravitreally to dystrophic rats, aged 1–18 weeks, survived 4 weeks in 80% of the animals. Takahashi et al. [13] also describe good survival after intravitreal transplantation of LacZ-transduced AHPCs to healthy immature (P0–P2) rat retinas. The present study shows a modest survival of transplanted cells compared with the results obtained from transplantation of AHPCs [13, 19].
Instead of an intravitreal grafting technique, we use a subretinal transplantation approach in order to obtain retinal integration of the cells into the adult retina. The surgical method, per se, causes a break of the outer blood-retinal barrier, the retinal pigment epithelium. This break triggers an inflammatory response towards the host retina as well as towards the engrafted precursor cells. From earlier experiments it is documented that transplantation with the RN33B cell line to adult retina only results in surviving cells if immunosuppression is used [17, 22].
Migration of Grafted Cells
An extensive migratory capacity of the RN33B cells has been shown after transplantation to different regions of the neonatal and adult rodent brain [29, 37–39]. As we reported recently, when RN33B cells are transplanted to the adult retina, they migrate radially from the subretinal space into the host retina as well as laterally within the retina. In addition, grafted cells are found from one eccentricity to the other in cryoprotected sections of the adult SD rat retinas up to 16 weeks post-transplantation [17]. A similar distribution pattern was observed in adult Royal College of Surgeons rats 6 weeks post-grafting [22]. In the present study, we show on whole-mounted retinas that the grafted cells are distributed over a large area (up to 68% of the entire retinal area) at 8 weeks post-transplantation. This again indicates that RN33B cells migrate to regions distant from the implantation site in the host retina, although a large fraction of the grafted cells were found at or close to the implantation site.
Two types of neural migration patterns for immature cells have been described in the brain: the radial glial and the tangential pattern [40]. The first type uses the columnar architecture of the brain as a scaffold. The second, also described as the chain migration type, is explained as immature neuronal cells climbing on each other forming cell tubules [33]. In the present study, an additional finding on four of the retinas 8 weeks post-grafting were the tubules of cells close to the implantation site, resembling the chain migration formation of immature cells found in the rostral migratory stream of the adult brain [33].
The retina is highly columnar, organized by the radial glial Müller cells [41]. One might speculate that the precursor cells climb on the radial glial cells when entering the retina from the subretinal space, and then within the retina using an unknown mechanism(s) for lateral migration. In the two main neurogenic regions (hippocampus and subventricular zone) in the adult rodent brain, and especially in the subventricular zone and rostral migratory stream, neuroblasts migrating in chain-like structures express specific markers, such as doublecortin, which is a microtubule-associated protein important for neuronal migration [33, 42]. Another marker for migrating neuronal progenitors is collapsing-response-mediated protein 4 (CRMP-4/Tuc-4), a molecule involved in axonal guidance [33, 43]. In the present study, by using GFP/doublecortin or GFP/CRMP-4 double immunohistochemistry we did not observe immunoreactivity to doublecortin or CRMP-4 on the retinal chain migration tubules or in other grafted cells (Anita Blixt Wojciechowski and Karin Warfvinge, unpublished observations).
Differentiation of Grafted Cells
The grafted cells had the capacity to differentiate into complex morphologies within the adult retina. Several studies on brain transplantation of the currently used cells have addressed the issue on the differentiation process, and different grafting paradigms have shown that they differentiate into region-specific neurons, such as hippocampal, cortical pyramidal, and striatum-specific medium-spiny projection neurons [29, 37, 39, 44]. In addition, RN33B cells have been found to extend appropriate axonal projections as revealed by retrograde tracing studies [37, 44]. Moreover, RN33B cells exhibit multipotential differentiation, i.e., into neurons, astrocytes, and oligodendrocytes, when transplanted into the neonatal rat brain [29]. Recent data using the GFP-marker gene and electrophysiological recordings demonstrate that grafted RN33B cells can become functionally integrated within host cortical neural circuits [44].
Our recent results have demonstrated that these cells do not differentiate into retinal cells, judging from the lack of expression of retinal neuronal markers, but into glial cells, mainly oligodendrocytes [16, 17, 22]. Here, GFP-expressing cells at 3 and 8 weeks displayed a wide range of morphologies, including glial- and neuronal-like profiles. The glial-like cells had slender cell bodies with long processes, while the neuronal-like cells were multipolar with branched processes exhibiting multiple spines (Fig. 6A and 6B). Comparison between the retinas 3 and 8 weeks after grafting demonstrated similar types of differentiated cells.
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CONCLUSION |
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Still little is known about the factors that influence precursor cell survival, proliferation, and differentiation upon retinal and brain transplantation, therefore multipotent precursor cell transplantation to the retina is a valuable tool in investigating these issues. The possibility to introduce genes ex vivo to modify the precursor cells to produce different factors necessary for the diseased host retina gives an additional perspective. This emerging science will clearly be useful to the obstacles involved in retinal reconstruction.
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ACKNOWLEDGMENT |
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FOOTNOTES |
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REFERENCES |
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