December 2003
Volume 44, Issue 12
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Retinal Cell Biology  |   December 2003
Enhanced Induction of RPE Lineage Markers in Pluripotent Neural Stem Cells Engrafted into the Adult Rat Subretinal Space
Author Affiliations
  • Volker Enzmann
    From the Department of Ophthalmology and Visual Sciences and the
  • Russell M. Howard
    Kentucky Spinal Cord Injury Research Center, Department of Neurological Surgery, University of Louisville, Louisville, Kentucky.
  • Yasuyuki Yamauchi
    From the Department of Ophthalmology and Visual Sciences and the
  • Scott R. Whittemore
    Kentucky Spinal Cord Injury Research Center, Department of Neurological Surgery, University of Louisville, Louisville, Kentucky.
  • Henry J. Kaplan
    From the Department of Ophthalmology and Visual Sciences and the
Investigative Ophthalmology & Visual Science December 2003, Vol.44, 5417-5422. doi:10.1167/iovs.03-0468
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      Volker Enzmann, Russell M. Howard, Yasuyuki Yamauchi, Scott R. Whittemore, Henry J. Kaplan; Enhanced Induction of RPE Lineage Markers in Pluripotent Neural Stem Cells Engrafted into the Adult Rat Subretinal Space. Invest. Ophthalmol. Vis. Sci. 2003;44(12):5417-5422. doi: 10.1167/iovs.03-0468.

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

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Abstract

purpose. To investigate the differentiation of rat neural stem cells (rNSCs) into cells of retinal pigment epithelial (RPE) lineage both in vitro and in vivo, after subretinal transplantation into normal rats and in a sodium iodate (NaIO3) model of RPE loss.

methods. rNSCs prepared from the cortex of embryonic day (E)14 Fisher F344 rats were cocultured with different concentrations of vasoactive intestinal peptide (VIP), adult rat RPE cells, or neurosensory retina (NSR) for 5 days. Cell morphology and expression of RPE-specific markers (cytokeratin, CD68, microphthalmia-inducing transcription factor [MITF]) were studied. Additional antibodies used to characterize the rNSCs were markers for stem cells (nestin), immature neurons (βIII-tubulin), astrocytes (glial fibrillary acidic protein [GFAP]), and oligodendrocytes (Rip). In in vivo studies, 106 green fluorescent protein [GFP]–labeled rNSCs were injected subretinally in either normal adult Lewis rats or NaIO3-treated rats (70 mg/mL NaIO3 administered intravenously 7 days before transplantation).

results. In vitro VIP-treated rNSCs changed from round cells to glia-like cells with processes that stained for both GFAP and nestin. In addition, small clusters of flattened, polygonal cells with an epithelial-cell–like shape that stained for cytokeratin and CD68 were observed. Coculture of rNSCs with RPE cells, but not with NSR, also led to cells of this phenotype. After transplantation, nestin+ and GFP+ rNSCs were visible subretinally as a transplant. In addition, more than 50% of transplanted rNSCs were cytokeratin+ and CD68+.

conclusions. Very few rNSCs differentiate in vitro into epithelial-like cells that express RPE-specific markers. In vivo, this differentiation is remarkably enhanced after subretinal engraftment. Thus, transplantation of NSCs into the subretinal space may be a therapy for retinal diseases involving an RPE abnormality.

Stem cells have the capacity to self-renew as well as to give rise to specialized cell types. They are uncommitted and remain uncommitted until they receive a signal to develop into distinct cell types. 1 Besides embryonic stem cells that are pluripotent and derived from the blastocyst, 2 adult stem or precursor cells, which have a more limited potency to give rise to specialized cells, are found in different somatic tissues including the central nervous system (CNS) (see review 3 ). Irrespective of their origins, stem and precursor cells are multipotent in differentiation and transdifferentiation, responding to stimuli both in vivo and in vitro. 4 5 6  
Neural stem cells, expressing the neural intermediate filament nestin, 7 are found in regions of continued high-rate neurogenesis-like dentate gyrus or subventricular zones throughout life. 8 They can grow in vitro in the presence of defined growth factors (e.g., basic fibroblast growth factor [bFGF] and epithelial growth factor [EGF] 9 ) and can be directed to differentiate into neuronal or glial cell types. 10 11 Furthermore, the microenvironment, healthy or pathophysiological, may play a pivotal role in the fate of the neural precursor cells after transplantation. 12 13 Neural stem cells appear to have a wide differentiation potential, in that it is possible to turn these cells into a range of blood cell types in vivo. 5 However, the question remains of whether this transdifferentiation is physiologic. At least two explanations for this phenomenon are plausible: first, there may be an opening of options for cells after relaxation of a geographical restraint (e.g., when brain cells are injected intravenously) or alternatively, a certain error rate in cell differentiation may occur. 14  
Retinal cells arise from multipotential progenitors that may provide a substrate for retinal regeneration. These retinal stem cells, found in low numbers in either the ciliary margin 15 or retina 16 17 give rise to neurons and glia, but few oligodendrocytes in vitro. Although these cells are capable of self-renewal and multilineage differentiation in vitro, they develop mostly into glial cells after engraftment into the adult retina. 18 During vertebrate eye development, the rise of the pigmented epithelium from the proximal optic vesicle is solely dependent on the microphthalmia-inducing transcription factor [MITF]. 19 Factors with regulatory function for MITF expression include VIP, which acts also as a differentiation promoter for a functional RPE cell monolayer and has trophic and mitogenic properties in embryonic neural tissue. 20 This 28-amino-acid peptide is found in the aqueous humor, choroid, retina (especially in amacrine cells), and the nerves of the uvea. 21 22 23  
The purpose of the present study was to investigate whether ectopic stem cells could be induced to adopt a RPE phenotype. The importance of this approach can be found in age-related macular degeneration (AMD), the leading cause of blindness in developed countries after age 55. 24 The major limiting feature in visual recovery after submacular surgery for exudative AMD is the unavoidable removal of RPE beneath the fovea at the time of surgery. Thus, the rapid repopulation of the bare area of Bruch’s membrane beneath the fovea by RPE is essential for recovery of central vision. Unfortunately, the removed RPE cells are not replaced by the proliferation of adjacent RPE cells. Thus, the simultaneous transplantation of RPE cells before irreversible atrophy of the foveal photoreceptors has occurred is a reasonable option. However, allogeneic adult RPE cells do not attach to senescent Bruch’s membrane efficiently and do not undergo proliferation and spreading to fill in the defect. 25 Furthermore, graft rejection may occur after allogeneic adult RPE transplantation. 26 27 Thus, we would like to study the ability of alternative pluripotent stem cells which have reduced immunogenicity 28 to accomplish this goal more effectively. 
Methods
Cell Cultures
rNSCs were prepared from embryonic day (E)14 Fisher 344 rats by dissection of the cortices, as described previously. 29 Briefly, the cerebral cortices were carefully dissected in L15 media and the meninges removed. The tissue was then gently triturated three times with a P1000 pipette. Chunks of tissue were allowed to settle down and the suspended, single cells (viabilities ranged between 60% and 80%) were plated on tissue culture dishes precoated with 15 μg/mL poly-l-ornithine and 1 μg/mL fibronectin and grown at 37°C in a 5% CO2 atmosphere. The cells were maintained in proliferation medium (DMEM/F-12 containing 20 ng/mL bFGF and N2 supplement). These proliferating cells stained with the neural stem cell marker nestin and did not express any markers of more mature astrocytes (glial fibrillary protein [GFAP]), oligodendrocytes (Rip, O4, galactocerebroside [GalC], myelin basic protein [MBP]) or neurons (microtubule protein 2 [Map2], neuron-specific βIII-tubulin, or 160-kDa neurofilament protein). 9 For the subretinal transplantations, passage 1 to 3 rNSCs (postnatal day [P]1–P3) were infected with green fluorescent protein (GFP) using a retroviral vector (pLZRSEGFP). 13  
The rat RPE cells were obtained from adult Lewis rat eyes (4–6 weeks old) by enzymatic digestion with 0.25% trypsin and 1 mM EDTA and 0.5% collagenase and 0.05% hyaluronidase for 1 hour each and cultured in DMEM, 10% FBS, and gentamicin. These cells were routinely stained with antibodies against a broad range of epidermal keratins (AE1/AE3; RDI, Flanders, NJ) for determination of their epithelial origin. P3 to P5 RPE cells and P1to P3 rNSCs were used. All cell culture material and chemicals were purchased from Invitrogen (Carlsbad, CA). 
Transplantation
Lewis rats, 3 to 4 weeks old, were used in all experiments and were purchased from Harlan Laboratories (Indianapolis, IN). The animals were treated according to the regulations in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and after approval of the protocol by the University of Louisville Institutional Animal Care and Use Committee (IACUC). A total of 10 eyes, n = 5 per group, were divided into an untreated group and a group injected intravenously with 2% NaIO3 (70 mg/kg; Sigma-Aldrich, St. Louis, MO) through the tail vein. In both groups rNSCs (105 cells/μL) were injected transclerally into the subretinal space of the superior hemisphere of the right eye using a glass micropipette. A microinjector infusion pump was used for injection of a 10-μL cell suspension. A scleral incision was made close to the superior limbus, the micropipette was then inserted into the scleral wound and the fluid was injected into the subretinal space at 1 μL/sec. The treated group received the rNSCs transplant 7 days after the NaIO3 injection. 
In Vitro Treatments
rNSCs were cultured in proliferating medium without fetal bovine serum (FBS) or in differentiating medium (DMEM/F-12/N2/1%FBS). Increasing concentrations of VIP (5 × 10−9 M, 5 × 10−8 M, 5 × 10−7 M) were used to treat the cells for 5 days and the medium, containing VIP, was changed once. In parallel experiments, the rNSCs were cocultured with RPE at several ratios (1:1; 1:5, or 1:10) or with rat NSR obtained from Lewis rats. In addition, different treatments were combined (e.g., 5 × 10−7 M VIP added to a coculture of RPE cells and rNSCs 1:1). In the coculture experiments, the respective cell types were separated with a membrane (PET [polyethylene terephthalate]; BD Biosciences, Franklin Lakes, NJ) or a cellulose nitrate membrane (Whatman, Maidstone, UK) that allowed only the circulation of soluble factors between the two components. 
All experiments were performed three times and results expressed as mean ± SD. Statistical significance (P ≤ 0.05) was determined by Student’s t-test. 
Immunocytochemistry
After treatment, rNSC cultures were washed in PBS and fixed in 4% paraformaldehyde (PFA) at room temperature (RT) for 15 minutes. Subsequently, nonspecific binding was blocked with normal sheep serum (Serotec, Raleigh, NC) at RT for 30 minutes. The primary antibodies used to characterize the cells after the different treatments (Table 1) were diluted in PBS with 0.1% Tween 20 and incubated for 60 minutes at RT. After the cells were washed three times for 5 minutes each in PBS with 0.1% Tween 20, the secondary antibodies (see below) were applied for 30 minutes. Subsequent to repeated washing, the number of cells in four random visual fields were counted and the number of positive cells used to calculate the percent positive. 
For staining of the paraffin-embedded sections, the rats were killed 7 days after transplantation and the transplant-engrafted eyes were enucleated. The specimens were fixed with 4% PFA at 4°C overnight and then embedded in paraffin. Consecutive sections (5 μm) of the transplantation area mounted on slides (Superfrost; Fisher Scientific, Pittsburgh, PA) were used for transplant localization after a hematoxylin-eosin (H&E) overview staining followed by immunohistochemistry for stem and RPE cell markers. Briefly, the specimens were deparaffinized and microwaved in buffer (citrate or EDTA) at 350 W for 10 minutes. Sections were then blocked with 3% normal serum (Serotec) corresponding to the animal in which the secondary antibody was raised at RT for 1 hour. The primary antibodies (Table 1) diluted in PBS with 0.1% Tween 20 (0.2% Triton X-100 for anti-GFP), were incubated overnight at 4°C. After the sections were washed three times for 5 minutes each at RT in PBS with 0.1% Tween 20, the secondary antibodies (described later) were applied for 1.5 hours. The sections were mounted in aqueous medium (Aqua Mount; Lerner, Pittsburgh, PA) after additional washing in PBS. 
Specific binding was visualized with secondary antibodies against mouse- IgG/rabbit-IgG conjugated with Cy3 (C2306/C2181; Sigma-Aldrich) or against chicken-IgG conjugated with FITC (AP162AF; Chemicon, Temecula, CA) and viewed under fluorescence optics (Olympus, Melville, NY). Cell morphology was characterized by phase-contrast microscopy and both RPE loss and the location of the transplants were documented with H&E staining. 
Results
In Vitro Experiments
The morphology of rNSCs after 5 days in culture changed from round without FBS (e.g., proliferating conditions; Fig. 1A ) to process-bearing in the presence of FBS (Fig. 1B) and VIP (Figs. 1C 1D) . However, clusters of flattened, polygonal cells were observed only after treatment with VIP (Fig. 1D , arrows) and coculture with RPE cells (data not shown). 
The stem cell origin of rNSCs was confirmed by nestin-positive staining (not shown). The number of nestin-positive cells ranged between 40.0% ± 1.5% and 73.7% ± 2.2%, depending on the culture conditions. The astrocyte marker GFAP was found on 37.6% ± 8.8% of rNSCs after VIP treatment, 48.0% ± 5.4% after coculture with adult RPE cells, and 69.0% ± 4.0% after coculture with NSR (Table 2) . Very few cells were positive for the oligodendrocyte marker Rip. The early neuronal marker βIII-tubulin was not detectable. There was no statistically significant difference between the groups. 
Clusters with cytokeratin-positive cells with epithelial-cell–like shape were seen after treatment with VIP (Fig. 1E) or coculture with adult RPE cells (not shown). Treatment with VIP resulted in the highest number of cytokeratin-positive cells (Table 2) , 0.1% ± 0.04% after 5 days, compared with only 0.05% ± 0.005% after coculture. Under the same conditions, we observed CD68-positive cells in these clusters (Fig. 1F) with VIP treatment resulting in 0.014% ± 0.007%–positive cells compared with 0.03% ± 0.004% after coculture. Increasing concentrations of VIP led to higher numbers of cytokeratin-positive as well as CD68-positive rNSCs (data not shown). 
Coculture of rNSCs with increasing ratios of RPE cells did not influence rNSC differentiation. Furthermore, a combination of both treatments (5 × 10−7 M VIP added to a coculture of RPE cells and rNSCs 1:1) did not show any additional effect. rNSCs cultured without treatment and in coculture with NSR or with 5 × 10−7 M VIP (for 5 days) without FBS showed no positive staining for RPE-lineage markers. 
In Vivo Experiments
Nestin-positive rNSCs were visible within the subretinal space 7 days after transplantation in both the normal and NaIO3-treated rats (Table 3) . Five of five rats receiving GFP-labeled rNSC grafts contained green fluorescence in the engrafted cells and in the other five recipients of the transplants were readily identifiable by their localization to a single area at the site of transplantation. In normal rats as well as NaIO3-treated recipients, transplanted stem cells stained positive for the RPE lineage-specific markers, cytokeratin and CD68 (Fig. 2) , with the exception of transplant 5 in which cytokeratin was not expressed. The host RPE monolayer was also both cytokeratin and CD68 positive. Five of the 10 transplants stained for the RPE marker MITF. Inflammation was noted only within transplant 5 on H&E sections. Those CD68-positive cells probably represent infiltrating macrophages. 
Treatment with NaIO3 led to the total loss of RPE after 7 days and resulted in negative staining for cytokeratin and CD68. However, transplanted rNSCs in these animals were nestin- and GFP-positive, and expressed the RPE-specific markers cytokeratin, CD68, and MITF (Fig. 3)
Discussion
In our in vitro experiments, we observed that rNSCs mainly differentiated into astrocytes, with very few oligodendrocytes and no neurons. These results differ from those previously reported after the removal of mitogen from rNSCs where they differentiated into all three cell types: astrocytes (32%), oligodendrocytes (15%), and neurons (13%). 29 In addition, a small number of cells, with a flat, epithelial-cell–like morphology stained positive for cytokeratin and CD68 after treatment with VIP or coculture with adult RPE cells. Cytokeratin is expressed by RPE cells in different vertebrate species and can be used for the identification of this cell type. 30 CD68 is also expressed on neuroectodermally derived RPE cells, as well as bone marrow–derived macrophages. 31 Thus, the presence of both cytokeratin and CD68 by the rNSCs suggests either the upregulation of RPE markers in the NSC or possibly partial transdifferentiation of the NSCs along the RPE lineage. We cannot distinguish between these possibilities at present. 
We did not explore the mechanism by which VIP or coculture with adult RPE cells resulted in the expression of these RPE-lineage markers. It is known that VIP receptors are widely distributed in embryonic neural tissues 32 and that VIP can upregulate MITF, a transcription factor essential for the development of RPE cells from neural progenitors in the eye. 19 It is likely that a soluble factor was also responsible for the effect observed after coculture with RPE cells, because the cells were separated by a filter. This effect was RPE specific, as coculture with NSR did not produce similar results. It is recognized that insulin-like growth factor (IGF)-1 is involved in the MITF signaling pathway 33 and can be produced by RPE cells. 34 However, the N2 supplement used by us to maintain the rNSCs contains 5 μg/mL of insulin, a concentration that should activate IGF-1 receptors and result in the expression of RPE-specific markers. Because we did not observe this, it is unlikely that the effect of RPE coculture is mediated by IGF-1. 
Unfortunately, the number of cells that expressed RPE markers after in vitro culture was very few and would not be sufficient to transplant and result in the repopulation of a denuded Bruch’s membrane. However, we observed a much higher number of rNSCs that expressed RPE-specific markers after transplantation into the subretinal space of both normal and NaIO3-treated rats. These results are consistent with previous reports of NSCs engrafted in both the normal and injured CNS. 35 In addition, during ontogenesis the various cell types of the retina, including the RPE, develop from neuroectoderm. 36 The positive staining of transplants for both cytokeratin and CD68 shows rNSCs are better able to upregulate the expression of RPE markers in vivo than in vitro, in both a normal environment and in an altered subretinal space (NaIO3). This enhanced RPE-like differentiation probably reflects the fact that the epigenetic cues necessary to induce the expression of the RPE-like phenotype that are missing in vitro are expressed in the subretinal space. In addition, both the normal and the NaIO3-altered subretinal spaces provide the microenvironment for survival of the transplanted stem cells. Similar results have been shown by Kale et al. 38 with the incorporation of transplanted bone marrow stem cells into normal and ischemically injured renal tubules. The sporadic staining observed for MITF may reflect different stages of differentiation of pigmented epithelial cells in the transplants, because MITF is involved in the late phase of this process. 38  
There has been considerable enthusiasm about the transdifferentiation potential of stem cells derived from various sources. CNS stem cells were shown to differentiate into blood cells 5 and skeletal muscle. 39 Bone marrow stem cells have been reported to transdifferentiate into mesenchymal cells, 40 brain, 41 heart, 42 skeletal muscle, 43 and liver. 44 However, recent data suggest that cell fusion may be responsible for some of these effects, 45 46 and experiments with highly purified precursor populations suggest that transdifferentiation, if it occurs at all, is a very rare event. 6 14 47 48  
Our in vitro and in vivo observations suggest that rNSCs may have the potential to replace lost RPE cells in nonexudative AMD, as well as in exudative AMD after submacular surgery. However, evidence for successful differentiation of engrafted stem cells requires demonstration of functional integration. Induction of only phenotypic markers is insufficient. In the case of RPE replacement, functional tests such as phagocytosis by the “transdifferentiated” NSC in vitro and restoration of normal electrophysiology and vision-dependent behavior in vivo are needed. Until such functional transdifferentiation is observed, stem cell grafting as a strategy to repair the damaged nervous system remains a promising but elusive goal. 
 
Table 1.
 
Antibodies Used for NSC Characterization
Table 1.
 
Antibodies Used for NSC Characterization
Antibody Company and Antibody Number Dilution Cell Type Identified
anti-Pan cytokeratin Sigma-Aldrich, St. Louis, MO; C2931 1:100 Epithelial cells (RPE)
anti-CD68 Bachem Bioscience, Philadelphia, PA, T3003 1:200 Macrophages, RPE
anti-Nestin DSHB Rat-401* 1:25 Undifferentiated NSC
anti-βIII-tubulin Covance, Vienna, VA; PRB-435P 1:100 Early neurons
anti-GFAP Sigma-Aldrich; G9269 1:200 Astrocytes
anti-Oligodendrocytes Chemicon, Temecula, CA; MAB1580 1:200 Oligodendrocytes
anti-GFP Chemicon; AB16901 1:100 GFP+ transplants
anti-MITF NeoMarkers, Fremont, CA; MS-771 1:50 RPE cells
Figure 1.
 
Culture of rNSCs (5 × 105 cells/well) for 5 days without FBS (A), with 1% FBS (B), and with 1% FBS+5 × 10−7 M VIP (C, D). Arrows: cell clusters with an epithelial shape. The latter cultures were cytokeratin (E) and CD68 (F) positive. Magnification: (A, D) ×50; (B, C, E, F) ×100.
Figure 1.
 
Culture of rNSCs (5 × 105 cells/well) for 5 days without FBS (A), with 1% FBS (B), and with 1% FBS+5 × 10−7 M VIP (C, D). Arrows: cell clusters with an epithelial shape. The latter cultures were cytokeratin (E) and CD68 (F) positive. Magnification: (A, D) ×50; (B, C, E, F) ×100.
Table 2.
 
Cell Types after 5-Day Culture of rNSCs under Different Conditions
Table 2.
 
Cell Types after 5-Day Culture of rNSCs under Different Conditions
Condition Nestin-Positive Cells CD68-Positive Cells Cytokeratin-Positive Cells Astrocytes Oligodendrocytes Neurons
Without serum 51.3 ± 7.7 None None ND ND ND
1% FBS 53.7 ± 7.5 None None ND ND ND
VIP (5× 10−7 M)+ 1% FBS 73.7 ± 2.2 0.014 ± 0.007 0.1 ± 0.04 36.7 ± 8.8 0.13 None
NSR+1% FBS 40.0 ± 1.5 None None 69.0 ± 4.0 2 Cells None
RPE+1% FBS 67.0 ± 6.2 0.03 ± 0.004 0.05 ± 0.005 48.0 ± 5.4 0.03 None
Table 3.
 
RPE Lineage–specific Markers after Subretinal Transplantation of rNSC into Normal or NaIO3-Treated Lewis Rats
Table 3.
 
RPE Lineage–specific Markers after Subretinal Transplantation of rNSC into Normal or NaIO3-Treated Lewis Rats
Transplant Treatment Transplant Nestin GFP Cytokeratin CD68 MITF
1 Subretinal + + +
2 Subretinal + + +
3 Subretinal + N/A + + +
4 Subretinal + N/A + +
5 Subretinal + N/A +
6 NaIO3 Subretinal/suprachoroidal + + + + +
7 NaIO3 Subretinal + + + + +
8 NaIO3 Subretinal + + + + +
9 NaIO3 Suprachoroidal + N/A + + +
10 NaIO3 Subretinal + N/A + +
Summary of results 9/10 Transplants successfully placed into the subretinal space 10/10 5/5 9/10 8/10 5/10
Figure 2.
 
Transplanted rNSCs into the normal subretinal space. Stained with H&E (A) or for nestin (B), cytokeratin (C), and CD68 (D); arrows: host RPE. Magnification: (A) ×50; (BD) ×100.
Figure 2.
 
Transplanted rNSCs into the normal subretinal space. Stained with H&E (A) or for nestin (B), cytokeratin (C), and CD68 (D); arrows: host RPE. Magnification: (A) ×50; (BD) ×100.
Figure 3.
 
Transplanted rNSCs into the altered subretinal space. Stained for GFP (A) and cytokeratin (B). (C) Merged image of (A) and (B); arrows: double-stained rNSCs. Magnification, ×40.
Figure 3.
 
Transplanted rNSCs into the altered subretinal space. Stained for GFP (A) and cytokeratin (B). (C) Merged image of (A) and (B); arrows: double-stained rNSCs. Magnification, ×40.
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Figure 1.
 
Culture of rNSCs (5 × 105 cells/well) for 5 days without FBS (A), with 1% FBS (B), and with 1% FBS+5 × 10−7 M VIP (C, D). Arrows: cell clusters with an epithelial shape. The latter cultures were cytokeratin (E) and CD68 (F) positive. Magnification: (A, D) ×50; (B, C, E, F) ×100.
Figure 1.
 
Culture of rNSCs (5 × 105 cells/well) for 5 days without FBS (A), with 1% FBS (B), and with 1% FBS+5 × 10−7 M VIP (C, D). Arrows: cell clusters with an epithelial shape. The latter cultures were cytokeratin (E) and CD68 (F) positive. Magnification: (A, D) ×50; (B, C, E, F) ×100.
Figure 2.
 
Transplanted rNSCs into the normal subretinal space. Stained with H&E (A) or for nestin (B), cytokeratin (C), and CD68 (D); arrows: host RPE. Magnification: (A) ×50; (BD) ×100.
Figure 2.
 
Transplanted rNSCs into the normal subretinal space. Stained with H&E (A) or for nestin (B), cytokeratin (C), and CD68 (D); arrows: host RPE. Magnification: (A) ×50; (BD) ×100.
Figure 3.
 
Transplanted rNSCs into the altered subretinal space. Stained for GFP (A) and cytokeratin (B). (C) Merged image of (A) and (B); arrows: double-stained rNSCs. Magnification, ×40.
Figure 3.
 
Transplanted rNSCs into the altered subretinal space. Stained for GFP (A) and cytokeratin (B). (C) Merged image of (A) and (B); arrows: double-stained rNSCs. Magnification, ×40.
Table 1.
 
Antibodies Used for NSC Characterization
Table 1.
 
Antibodies Used for NSC Characterization
Antibody Company and Antibody Number Dilution Cell Type Identified
anti-Pan cytokeratin Sigma-Aldrich, St. Louis, MO; C2931 1:100 Epithelial cells (RPE)
anti-CD68 Bachem Bioscience, Philadelphia, PA, T3003 1:200 Macrophages, RPE
anti-Nestin DSHB Rat-401* 1:25 Undifferentiated NSC
anti-βIII-tubulin Covance, Vienna, VA; PRB-435P 1:100 Early neurons
anti-GFAP Sigma-Aldrich; G9269 1:200 Astrocytes
anti-Oligodendrocytes Chemicon, Temecula, CA; MAB1580 1:200 Oligodendrocytes
anti-GFP Chemicon; AB16901 1:100 GFP+ transplants
anti-MITF NeoMarkers, Fremont, CA; MS-771 1:50 RPE cells
Table 2.
 
Cell Types after 5-Day Culture of rNSCs under Different Conditions
Table 2.
 
Cell Types after 5-Day Culture of rNSCs under Different Conditions
Condition Nestin-Positive Cells CD68-Positive Cells Cytokeratin-Positive Cells Astrocytes Oligodendrocytes Neurons
Without serum 51.3 ± 7.7 None None ND ND ND
1% FBS 53.7 ± 7.5 None None ND ND ND
VIP (5× 10−7 M)+ 1% FBS 73.7 ± 2.2 0.014 ± 0.007 0.1 ± 0.04 36.7 ± 8.8 0.13 None
NSR+1% FBS 40.0 ± 1.5 None None 69.0 ± 4.0 2 Cells None
RPE+1% FBS 67.0 ± 6.2 0.03 ± 0.004 0.05 ± 0.005 48.0 ± 5.4 0.03 None
Table 3.
 
RPE Lineage–specific Markers after Subretinal Transplantation of rNSC into Normal or NaIO3-Treated Lewis Rats
Table 3.
 
RPE Lineage–specific Markers after Subretinal Transplantation of rNSC into Normal or NaIO3-Treated Lewis Rats
Transplant Treatment Transplant Nestin GFP Cytokeratin CD68 MITF
1 Subretinal + + +
2 Subretinal + + +
3 Subretinal + N/A + + +
4 Subretinal + N/A + +
5 Subretinal + N/A +
6 NaIO3 Subretinal/suprachoroidal + + + + +
7 NaIO3 Subretinal + + + + +
8 NaIO3 Subretinal + + + + +
9 NaIO3 Suprachoroidal + N/A + + +
10 NaIO3 Subretinal + N/A + +
Summary of results 9/10 Transplants successfully placed into the subretinal space 10/10 5/5 9/10 8/10 5/10
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