Cover Page
The handle http://hdl.handle.net/1887/19856 holds various files of this Leiden University
dissertation.
Author: Cogliati, Tiziana Paola
Title: Study and retina allotransplantation of porcine ciliary epithelium (CE)-derived cells
Date: 2012-09-27
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Chapter 5
Molecular Vision 2011; 17:2580-2595 <http://www.molvis.org/molvis/v17/a279>
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TABLE 2. PRIMARY ANTIBODIES USED FOR IMMUNOHISTOCHEMICAL ANALYSIS.
Antibody
Recoverin
Rhodopsin (Rho4D2)
‹Œ
Calbindin
RPE65
Ki67
_
HuC/D
Host
rabbit
mouse
mouse
rabbit
mouse
mouse
mouse
mouse
Dilution
1:1000
1:100
1:400
1:1500
1:400
1:300
1:350
1:200
Source
Kind gift from Karl-Wilhelm Koch
‹~
Sigma-Aldrich
$
$
BD Biosciences
Sigma-Aldrich
$`
Host animal, dilution and source for each antibody are shown.
in 10% NGS, 0.3% Triton X-100, and 0.01% NaN3 in PBS for
1 h at room temperature, followed by overnight incubation at
4 ´# The slides were incubated for 24 h at 4 ´ with the
primary antibody diluted in 10% NGS, 0.3% Triton X-100,
and 0.01% NaN3 in PBS. For double labeling, the second
primary antibody was added after removal of the first primary
antibody and was incubated for another 24 h at 4 ´# After
removal of the second primary antibody, the slides were
washed for 6×5 min with PBS and were incubated in the first
secondary antibody (Alexa _–££ goat antimouse) diluted
1:500 in PBS for 1 h. Subsequently, incubation with another
secondary antibody (Alexa _„ƒ£ goat antirabbit), was
performed for 1 h. The slides were washed for 3×5 min with
PBS and were counterstained and mounted as described
above. The cells were visualized and the images were captured
with an epifluorescence microscope (Nikon) using Nis
Elements (Nikon) software. The number of positive cells was
counted in 20 random fields at 40× magnification.
washed in PBS. After removal of the cornea and lens, the eyes
were fixed in 4% PFA in PBS for 1 h at room temperature.
The eyecups were cryoprotected in 10% sucrose for 6 h
followed by 30% sucrose overnight, embedded in an optimal
cutting temperature (OCT) compound (Sakura, Kobe, Japan),
and were snap frozen in an isopentane bath on dry ice.
Transverse cryosections (20 μm) were cut, mounted onto
Superfrost Plus glass slides (Fisher Scientific, Loughborough,
›‹$
½£‰´#
Immunohistochemistry: Immunohistochemistry on tissue
sections was performed as described previously [ˆ£—# Briefly,
slides were thawed at room temperature and were post-fixed
in 4% formaldehyde (Sigma-Aldrich) in PBS for 20 min at
room temperature. After rinsing in PBS, sections were
blocked for 1 h in 10% normal goat serum (NGS), 0.3% Triton
X-100, 0.01% NaN3 in PBS, at room temperature. Slides were
Ÿ–
–´!
10% NGS, 0.3% Triton X-100, and 0.01% NaN3 in PBS. The
primary antibodies used are listed in Table 2. After removal
of the primary antibody, slides were washed 6×5 min in PBS
and were incubated for 1 h at room temperature in a secondary
antibody (Alexa Fluor–££ goat anti-mouse or goat anti-rabbit),
1:500 in PBS. After 3×5 min washing steps in PBS, cell nuclei
were counterstained with 5 ¸ DAPI (Invitrogen) for 10 min.
The slides were mounted in a fluorescent mounting medium
(Dako, Ely, UK). Negative immunohistochemistry controls
were performed in parallel by omission of the primary
antibody. Immunoreactive cells were visualized and images
were recorded using an inverted confocal microscope (Nikon,
Eclipse TE 2000-U, Tokyo, Japan) and Nikon EZ-C1
software. Every tenth or twentieth section (200–400 μm step)
was stained for the same antibody.
RESULTS
Analysis of the gene expression of CE-derived spheres:
Expression of the key pluripotency genes [Ÿ¥$Ÿ–— and the
genes active during normal retinal development was analyzed
by RT–PCR using RNA extracted from P1 CE-derived
spheres. Transcripts for three pluripotency genes, namely
cMyc, Klf4, and Sox2 were present in CE-derived cultures,
while mRNAs for Nanog and Oct4 were not detected (Figure
1A). Transcription factors associated with the eye
specification and retinal histogenesis, including Six3, $
Hes1, Otx2 and Chx10, were also expressed in CE-derived
spheres (Figure 1B).
In vitro differentiation of CE-derived spheres: The capacity
of CE-derived cells from newborn pigs to differentiate into
retinal phenotypes was first evaluated in vitro, after plating
CE spheres on adherent substrates (poly-D-lysine and
laminin) and culturing for 20 days with a differentiation
medium containing serum and growth factors. Growth factors
(10 ng/ml bFGF and EGF) enhanced retinal differentiation in
the presence of 1% serum (Figure 2). Photoreceptor markers
For isolectin B4 staining, sections were blocked in 5%
BSA for 30 min, incubated with biothynilated Griffonia
simplicifolia Isolectin B4 (Vector) 1:100 for 1 h, washed for
3×5 min with PBS, and were finally incubated with
streptavidin-FITC 1:200 for 1 h.
For the immunocytochemistry of the differentiated cells,
post-fixation glass slides were washed 3× in PBS, incubated
Ÿ„£¥
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Chapter 5
Molecular Vision 2011; 17:2580-2595 <http://www.molvis.org/molvis/v17/a279>
© 2011 Molecular Vision
Figure 2. Microphotographs of the immunolabeling of newborn pig ciliary epithelium (CE)-derived cells after in vitro differentiation on polyD-Lysine, laminin coated coverslips in the presence of 1% serum and 10 ng/ml basic fibroblast growth factor (bFGF) and epidermal growth
factor (EGF). The images were acquired using an epifluorescent microscope. A, B: Cells labeled for recoverin are clustered together (arrows).
C: 4',6-diamidino-2-phenylindole (DAPI) nuclear staining corresponding to A and B. D, E: Cells double-labeled for recoverin (red, D) and
rhodopsin (green, D and E) are depicted by arrows. The focus is set to show rhodopsin-positive cell processes. Strong recoverin staining in
the cytoplasm masks the nuclear DAPI staining, which is shown separately in F. Cells positive for protein kinase Œ(‹Œ¡ green in G and
H, arrows) did not co-label with recoverin (red in G and H and another example in the inset in G, arrowheads). The focus is set to show the
processes of ‹Œ
cells in G and H, and the recoverin-labeled processes in the inset in G. Corresponding DAPI nuclear stain is shown
in I. J-K: A calbindin immunopositive cell is depicted by the arrow. The focus is set to show the processes of the labeled cell. Corresponding
DAPI nuclear stain is shown in L. M, N: Recoverin-positive cells (arrows in M and N) that had retained pigmented granules (arrow in the
bright-field image in O). P, Q: RPE-65 immunopositive cells (arrows) and the corresponding nuclear DAPI staining in R.
2585
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Chapter 5
Molecular Vision 2011; 17:2580-2595 <http://www.molvis.org/molvis/v17/a279>
© 2011 Molecular Vision
Figure 7. The immunoreactivity of transplanted ciliary epithelium (CE)-derived cells in the neuroretina. A: Microphotograph (1 . m confocal
stack) showing red CM-DiI-labeled cells positive for recoverin (green, arrows) in the outer nuclear layer ( L). B: Recoverin-only labeling
of the same confocal stack as in A. The arrows point to the transplanted cells. C: A 0.7 m confocal slice from the boxed area in A showing
double-labeled CM-DiI/recoverin positive cells (arrows) D: Green recoverin labeling in the same area (arrow). E: Red CM-DiI labeling and
4',6-diamidino-2-phenylindole (DAPI) nuclear staining of the same area. F, G: CM-DiI-labeled, protein kinase Œ (‹Œ cell (green)
in the inner plexiform layer (arrows). H, I: uC/D (green) and CM-DiI-positive cell in the ganglion cell layer (GCL arrow in H and I). J,
K: Calbindin (green) and CM-DiI-labeled cells in the GCL (arrows). The inset is at a higher magnification with visible processes (arrowhead).
The nuclei are labeled with DAPI (blue). Inner nuclear layer (I L).
2590
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