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Glaucoma
Identification of Adult Stem Cells in Schwalbe’s Line
Region of the Primate Eye
Barbara M. Braunger,
1
Bahar Ademoglu,
1
Sebastian E. Koschade,
1
Rudolf Fuchshofer,
1
B’Ann T. Gabelt,
2
Julie A. Kiland,
2
Elizabeth A. Hennes-Beann,
2
Kevin G. Brunner,
3
Paul L. Kaufman,
2
and Ernst R. Tamm
1
1
Institute of Human Anatomy and Embryology, University of Regensburg, Regensburg, Germany
2
Department of Ophthalmology & Visual Sciences, University of Wisconsin School of Medicine & Public Health, Madison,
Wisconsin, United States
3
Wisconsin National Primate Research Center, University of Wisconsin, Madison, Wisconsin, United States
Correspondence: Ernst R. Tamm,
Institute of Human Anatomy and
Embryology, University of Regens-
burg, Universit¨atsstr. 31, D-93053
Regensburg, Ger many;
ernst.tamm@vkl.uni-regensburg.de.
Submitted: May 25, 2014
Accepted: October 6, 2014
Citation: Braunger BM, Ademoglu B,
Koschade SE, et al. Identification of
adult stem cells in Schwalbe’s line
region of the primate eye. Invest
Ophthalmol Vis Sci. 2014;55:7499–
7507. DOI:10.1167/iovs.14-14872
PURPOSE.To identify stem cells in the chamber angle of the monkey eye by detection of 5-
bromo-20-deoxyuridine (BrdU) long-term retention.
METHODS.Four cynomolgus monkeys were treated with BrdU via subcutaneous pumps for 4
weeks. The eyes of two animals were processed immediately thereafter (group 1) while in the
other animals, BrdU treatment was discontinued for 4 weeks to allow identification of cells
with long-term BrdU retention (group 2). The number of BrdU-positive nuclei was quantified,
and the cells were characterized by immunohistochemistry and transmission electron
microscopy (TEM).
RESULTS.The number of BrdU-positive cells was higher at Schwalbe’s line covering the
peripheral end of Descemet’s membrane than in Schlemm’s canal (SC) endothelium,
trabecular meshwork (TM), and scleral spur (SS). Labeling with BrdU in SC, TM, and SS was
less intense and the number of labeled cells was smaller in group 2 than in group 1. In
contrast, in cells of Schwalbe’s line the intensity of BrdU staining and the number of BrdU-
positive cells was similar when group 1 and 2 monkeys were compared with each other,
indicating long-term BrdU retention. Cells that were BrdU-positive in Schwalbe’s line region
stained for the stem cell marker OCT4. Details of a stem cell niche in Schwalbe’s line region
were identified by TEM.
CONCLUSIONS.We provide evidence for a niche in the Schwalbe’s line region harboring cells
with long-term BrdU retention and OCT4 immunoreactivity. The cells likely constitute a
population of adult stem cells with the capability to compensate for the loss of TM and/or
corneal endothelial cells.
Keywords: Schwalbe’s line, stem cells, BrdU retention, monkey eye
The cells of the conventional or trabecular outflow
pathways, trabecular meshwork (TM) cells and Schlemm’s
canal (SC) endothelial cells are under constant mechanical
stress or strain. A major contributing factor to mechanical load
in the trabecular outflow pathways is the immediate neighbor-
hood of the ciliary muscle and its anterior tendons that connect
with the posterior parts of the trabecular meshwork.
1,2
Experimental studies using cholinergic drugs identified a
considerable stretch and distension of the trabecular meshwork
outflow pathways during ciliary muscle contraction,
3–5
a
scenario that likely occurs constantly during a lifetime while
the ciliary muscle contracts in accommodation. Another
mechanical factor acting in the aqueous humor outflow
pathways is the passage of aqueous humor. In response to
aqueous humor flow, cells of the SC inner wall partially detach
from their underlying extracellular matrix to form characteristic
outpouchings into the lumen of SC that have been termed giant
vacuoles.
1
It seems reasonable to assume that mechanical stress
or strain continuously leads to the loss of individual TM or SC
cells that need to be replaced in order to guarantee continuous
function of the trabecular outflow pathways. At least for the
TM, the replacement appears not to be sufficient over the
lifetime as a continuous loss of TM cells at a constant rate of
0.56% per year
6,7
or at a loss rate of 6000 TM cells per year
8
has
been shown. The loss of TM cells is higher in the TM of patients
with primary open-angle glaucoma (POAG), a factor that has
been hypothesized to cause or contribute to the increase in
resistance of the trabecular outflow pathways, and to the idea
of POAG as a consequence of aging.
6
Throughout the body, cells that have been lost because of
wear and tear can be replaced by mitosis of neighboring
differentiated cells. Indeed, results from autoradiographic
studies using
3
H-thymidine labeling indicated a slow ongoing
rate of DNA synthesis and presumably trabecular cell replica-
tion in the TM of the normal eye. In vivo studies showed a 0.1%
to 0.4% baseline incorporation rate in the TM of cynomolgus
monkeys,
9
and a higher (0.82% to 2.17%) in the TM of cats.
10
In
a study of organ-cultured human eyes, incorporation rates in
TM cells of 0.34% to 0.44% were found in control eyes and of
0.59% in laser-treated fellow eyes.
11,12
Alternatively, lost cells may be replaced by proliferation and
subsequent migration of adult stem cells that typically reside in
Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.
www.iovs.org jISSN: 1552-5783 7499
an adjacent niche. The stem cell niche is thought to constitute
an instructive or permissive environment by expressing certain
growth factors and/or extracellular matrix molecules.
13,14
Adult stem cells are usually defined as proliferative cells that
maintain their own numbers (self-maintenance) while dividing
a large number of times during which they can produce
daughter cells that are capable of differentiating down various
lineages (pluripotency).
15
Stem cells can also alter their self-
maintenance probability to ensure an expansion of stem cell
numbers if required following injury (clonogenic capacity).
Adult stem cells that are responsible for the maintenance of
tissue integrity and cell renewal throughout adulthood have
been described for various organs and tissues such as the
intestinal epithelium, the epidermis, the corneal epithelium
and the hematopoietic system.
16–21
In all these tissues, there is
a relatively high basic cellular turnover. More recently, adult
stem cells were also identified in brain
22,23
and retina
24
where
cell division is extremely rare and the situation is more
comparable to that in the TM.
There is evidence that adult stem cells reside somewhere in
the trabecular meshwork outflow pathways. Cells that
expressed stem cell markers were detected in cell cultures
that were initiated following isolation from fresh human TM by
fluorescence-activated cell sorting.
25
Moreover, primary cul-
tures initiated from human trabecular meshwork contain
relatively undifferentiated or progenitor cells which are
capable of forming spherical clusters or free-floating spheres
that may contain undifferentiated multipotent progenitor
cells.
26
However, it is unclear if adult TM stem cells exist in
vivo or where they have their niche in the anterior eye. In
general, adult stem cells are difficult to identify in vivo, because
they usually express only a limited amount of cell-specific
markers. A well-established noninvasive method to identify
stem cells monitors the incorporation of the nucleotide
analogue 5-bromo-20-deoxyuridine (BrdU) into the DNA of
cycling cells. Under steady-state conditions in vivo, most stem
cell populations are believed to divide infrequently and to have
a long cell cycle time. Murine hematopoietic stem cells, for
example, have been shown to slowly divide over a period of
1.5 to 3.0 months.
27
Quite similarly, epithelial stem cells in the
skin rarely divide within their niche but change properties
abruptly when stimulated to exit.
28
Hence, stem cells in the S-
phase that have incorporated BrdU will retain it over many
weeks, in contrast to more rapidly dividing cells, in which
BrdU becomes diluted over time.
Here we provide evidence that cells with long-term BrdU
retention reside in close association with the trabecular
outflow pathways in the eyes of cynomolgus monkeys. The
cells are localized at the peripheral end of Descemet’s
membrane in region of the Schwalbe’s line and stain for the
stem cell marker OCT4. Our results provide essential support
for the concept that Schwalbe’s line constitutes the niche for
adult TM stem cells in the primate eye.
MATERIALS AND METHODS
Animals and Treatment
Four cynomolgus monkeys (Macaca fascicularis) and two
rhesus monkeys (Macaca mulatta) that were housed at the
Wisconsin National Primate Research Center were used for the
present study. Rhesus monkeys were used for transmission
electron microscopy (see below). We administered BrdU
(Sigma-Aldrich Corp., St. Louis, MO, USA) to the four
cynomolgus monkeys via subcutaneous minipumps (ALZET
model 2ML4, ideally 1 pump/0.75 kg body weight; DURECT
Corp., Cupertino, CA, USA) in order to yield a rate of 8 mg/kg/d
(2.65 lL/h/pump) for 4 weeks. Pumps were removed from
animals that had BrdU treatment discontinued. Two animals
were killed immediately thereafter. In the other two animals,
BrdU treatment was discontinued for another 4 weeks before
they were killed (Fig. 1A). Animals were fixed under deep
anesthesia (intramuscular ketamine 25 mg/kg followed by
intravenous [IV] pentobarbital Na 15 mg/kg) by perfusion
fixation via the heart. Whole body exsanguination (resulting in
death) and fixation was accomplished by cannulating the left
ventricle of the heart, starting the infusion, then cutting the
right atrium to allow blood and fluid to escape. An entire liter of
0.1 M PBS was perfused through the system resulting in all the
blood being removed. Then the fixative solution (1 L 4%
paraformaldehyde/0.1 M PBS) was allowed to perfuse through.
FIGURE 1. Experimental setup and data analysis. (A) Schematic of the
BrdU treatment protocol. (B,C)1-lm semi-thin section through the
chamber angle of a monkey eye (B) and schematic drawing (C) of the
different regions that were analyzed for presence of BrdU-positive cells.
Schwalbe’s Line Stem Cells IOVS jNovember 2014 jVol. 55 jNo. 11 j7500
After perfusion, the 12:00 limbus was marked with a suture, the
eyes were enucleated, immersed in 4% PFA, and sent to
Germany for further analysis. A window was cut in the cornea
after enucleation to allow the fixative to penetrate. All
experiments were conducted in compliance with the ARVO
Statement on the Use of Animals in Ophthalmic and Vision
Research and institutional guidelines.
Immunohistochemistry
Eyes were dissected into quadrants (superior, inferior, tempo-
ral, and nasal) and embedded in paraffin. Paraffin sections at a
thickness of 6 lm were dewaxed, washed for 5 minutes each
in H
2
O and phosphate buffer (PhP), blocked with 5% dry milk
in 0.1 M PhP for 45 minutes, and incubated with primary
antibodies (BrdU 1:50; Invitrogen, Life Technologies, Darm-
stadt, Germany; CD31 1:100; Dako Deutschland GmbH,
Hamburg, Germany; OCT4 1:50; Santa Cruz Biotechnology,
Dallas, TX, USA) in 0.5% dry milk at 48C overnight. After three
washes in PhP (5 minutes each), secondary antibodies (anti-
mouse biotinylated 1:500 [Vector Laboratories, Inc., Burlin-
game, CA, USA] followed by Streptavidin AlexaFluor 488
1:1000 [Invitrogen] for BrdU, anti-rabbit AlexaFluor 546
[Invitrogen] 1:1000 for CD31 and OCT4) were applied for 1
hour at room temperature. If required, double staining with
antibodies against CD31 or OCT4 was performed following
counterstaining of cell nuclei with DAPI (Vectashield; Vector
Laboratories, Inc.) diluted 1:10 in fluorescent mounting
medium (Serva Electrophoresis GmbH, Heidelberg, Germany).
The sections were analyzed using a fluorescence light
microscope (AxioVision; Carl Zeiss Meditec MicroImaging
GmbH, Jena, Germany) and the appropriate software (AxioVi-
sion 4.8).
Quantification of BrdU-Positive Cells
Cells that were BrdU-positive were counted with respect to
their localization in the four quadrants of the eye and the
different subareas of the trabecular outflow pathways (Fig. 1).
For subareas, we selected the different regions of the TM; uveal
and corneoscleral TM, juxtacanalicular tissue (JCT), scleral spur,
and the endothelial layer of SC (Figs. 1B, 1C). In addition, we
counted cells in the most anterior, nonfiltering part of the TM
localized close to Schwalbe’s line and the transition zone to the
cornea. This region is known in the cynomolgus and rhesus
monkey as ‘‘operculum.’’ We distinguished operculum cells
(i.e., cells that are underneath the peripheral end of Descemet’s
membrane), from Schwalbe’s line cells (i.e., cells that reside on
the inner surface of Descemet’s membrane). The number of
BrdU-positive cells was normalized to the number of DAPI-
positive cells in the respective quadrant, in the respective
subarea of the trabecular meshwork outflow pathways or in the
Schwalbe’s line region. A minimum of 15 different sections was
analyzed per quadrant. To avoid double counting of BrdU-
positive cells, sections were separated by at least 12 lm.
Possible differences between the four quadrants were tested
with a linear mixed regression model accounting for the
nonindependence of sections from the same region and eye,
and of sections from differing regions within the same eye (see
‘‘Statistics’’ section). For the data presentation in the diagrams,
we calculated the mean value of these 15 or more different
sections per quadrant for all eyes of the treatment group (as
defined in Fig. 1A). Possible differences between the different
subareas of the chamber angle outflow pathway were again
analyzed using a linear mixed regression model (see ‘‘Statistics’’
section). For the data presentation in the diagrams, the mean
value for each subarea (as defined in Fig. 1C) across all eyes per
group (as defined in Fig. 1A) is shown.
Transmission Electron Microscopy
For transmission electron microscopy (TEM), untreated eyes
from two rhesus monkeys (Macaca mulatta)aged19to20years
were studied. The contralateral eye had been surgically treated
by viscocanalostomy.
29
The anterior chambers of each monkey
were exchanged with cationic 5 nm and noncationized 10 nm
gold solution at an intraocular pressure of ~15 mm Hg, and then
perfused at 25 mm Hg with Ito’s solution
30
from an elevated
reservoir. Under deep general anesthesia with IV pentobarbital
sodium, 15 mg/kg, these animalswere then perfused through the
heart with phosphate buffered saline, 0.1 mol/L (pH 7.4)
followed by Ito’s solution. The eyes were enucleated, windows
were cut in the cornea and sclera, and the eyes placed in the
same fixative and sent to Germany for electron microscopy.
Upon arrival, the eyes were placed in cacodylate buffer (pH 7.4)
for 24 hours to wash out fixative. Each eye was bisected and the
anterior halves were cut into quadrants by meridional sectioning.
Each quadrant was further dissected into wedge-shaped speci-
mens of 1- to 1.2-mm width that contained trabecular meshwork,
ciliary muscle, iris and adjacent cornea and sclera. All wedges
were dehydrated in ascending concentrations of alcohol and
embedded in epoxy resin according to standard protocols. A
least three specimens from each quadrant were analyzed. All
semi-thin sections were stained with Richardson’s stain
31
and
examined by light microscopy. Subsequently, meridional and
equatorial ultrathin sections were cut from each specimen that
had been investigated by light microscopy and stained with lead
citrate and uranyl acetate for TEM.
Statistics
All results are expressed as mean 6SEM. We used R 3.0.3
32
and lme4 1.1.7
33
to perform a linear mixed effects analysis of
the relationship between the BrdU-positive cell count
normalized to the total number of DAPI-stained cells and
quadrant location (super, inferior, nasal, temporal). Quadrant
location was specified as a fixed effect. As random effects,
intercepts for eyes and random slopes for quadrants per eye
were entered. Residual plots and quantile-quantile plots were
visually inspected to confirm homoscedasticity and normality
of residuals across groups. Statistical (P) values for the main
effect of quadrant were obtained by likelihood ratio testing of
the full model against the model without the fixed effect of
quadrant. Analyses were separately conducted for groups 1
and 2.
A similar linear mixed effects analysis was performed to
analyze the relationship between the BrdU-positive cell count
normalized to the total number of DAPI-stained cells and
trabecular meshwork regions or the chamber angle outflow
pathways (trabecular meshwork and Schwalbe’s line), respec-
tively. Analyses were again conducted separately for group 1
and 2 animals. Chamber angle outflow pathway localization
was specified as a fixed effect, and random intercepts for
localizations nested in eyes were specified. It was not possible
to expand the random effects structure to include random
slopes due to model convergence failure. Statistical values for
the main effect of chamber angle outflow pathway were
obtained by likelihood ratio testing of the full model against the
model without the fixed effect of chamber angle outfl ow
pathway localizations. The Kenward-Roger approximation was
used to calculate approximate degrees of freedom
34,35
and P
values for all pairwise comparisons were obtained from the t-
distribution with approximated degrees of freedom. Bonferro-
ni’s post hoc adjustment to Pvalues was used to control the
family-wise error rate. Values of P0.05 were considered to
be statistically significant.
Schwalbe’s Line Stem Cells IOVS jNovember 2014 jVol. 55 jNo. 11 j7501
RESULTS
We used four cynomolgus monkeys (Macaca fascicularis)to
identify adult stem cells in the trabecular meshwork outfl ow
pathways. To this end, BrdU was administered via subcutane-
ous minipumps for 4 weeks. Two animals were killed
immediately thereafter (group 1, chronic BrdU). In the two
other animals, BrdU treatment was discontinued for another 4
weeks before they were killed (group 2, chronic BrdU and
long-term retention; Fig. 1A). When sections through the
chamber angle were labeled for BrdU, positively stained nuclei
were regularly observed in the different regions of the
trabecular outflow pathways (Fig. 2A). Quantitative analysis
showed no significant preference in the number of BrdU-
positive cells for the different quadrants of the eyes (Fig. 2B).
This was true for both groups of monkeys. We next
distinguished BrdU-positive cells with regard to their specific
location in the TM outflow pathways and observed positively
labeled cells in all the different regions that were investigated,
(e.g., SC endothelium, JCT, corneoscleral and uveal TM, scleral
spur, and operculum; Fig. 3A). In the two monkeys of group 2
(chronic BrdU and long-term retention), the number of BrdU-
positive cells in the different regions was smaller than in group
1 (chronic BrdU). The highest number of BrdU-positive cells in
the eyes of group 2 was observed in SC endothelium, in which
the number of BrdU-positive cells was significantly higher than
in JCT, corneoscleral TM, and corneoscleral and uveal TM. Very
few BrdU-positive cells were observed in the scleral spur and
operculum of group 1 monkeys, while no positive cells were
observed in group 2 monkeys (Fig. 3B).
Next we performed double immunohistochemistry to
identify the nature of BrdU-stained cells. All BrdU-labeled cells
in the SC endothelial layer stained for CD31, a marker for
differentiated vascular endothelium (Fig. 3C). In contrast, SC
BrdU-positive cells did not react with antibodies against
octamer-binding transcription factor 4 (OCT4),
36
a homeodo-
main transcription factor that is critically involved in the self-
renewal of stem cells (Fig. 3D). Some highly reproducible, non-
nuclear and presumably extracellular OCT4 labeling was
observed in the JCT, which we regarded as nonstem cell
relevant since OCT4 is a transcription factor that localizes to
the nucleus to serve its function (Fig. 3D). Noteworthy, similar
to nuclei of SC cells, BrdU-positive nuclei in the different
regions of the TM outfl ow pathways were not immunoreactive
for OCT4.
We next turned our attention to Schwalbe’s line cells that
cover the peripheral end of Descemet’s membrane and which
do not constitute an anatomic part of the TM outflow
pathways. The relative number of BrdU-positive cells in this
area was significantly higher than among the cells of all the
different regions of the TM outflow pathways in both group 1
and 2 monkeys (Figs. 4A, 4B). We observed no difference in the
relative number of BrdU-labeled Schwalbe’s line cells between
groups 1 and 2 (Figs. 4A, 4B), a finding that strongly indicated
long-term BrdU retention. Double immunohistochemistry
showed that all BrdU-positive Schwalbe’s line cells were
immunoreactive for the stem cell marker OCT4 (Fig. 4C).
Some nuclei in the operculum area also stained for OCT4 (Fig.
4C).
Finally, we investigated by light and electron microscopy
the area of Schwalbe’s line region in which we had previously
observed cells with long-term BrdU retention and OCT4
immunoreactivity. Since the fixation protocol that had been
used for BrdU detection did not allow preservation of
ultrastructural details, we used untreated eyes from two rhesus
monkeys that had been fixed for TEM studies. In the area close
to the peripheral end of Descemet’s membrane, where most of
the BrdU/OCT4-positive cells reside (Fig. 5A), we regularly
observed cuboidal epithelial cells that differed in shape from
the flat adjacent corneal endothelial cells (Fig. 5B). The cells
frequently formed small clusters that were embedded in
furrows of Descemet’s membrane, a finding that was most
obvious when equatorial (frontal) sections were studied (Fig.
5C). In places, the cells completely filled gaps in Descemet’s
membrane and were in direct contact on their basal side with
TM cells from the nonfiltering operculum region of the TM
(Figs. 6A, 6B). Most of the cells in the Schwalbe’s line region
were characterized by the presence of numerous mitochondria
of the tubular type (Fig. 6C). In addition, we frequently
observed smaller cells with considerably less cytoplasm than
the mitochondria-rich cell type. The smaller cells were
typically engulfed by a mitochondria-rich cell (Fig. 6D).
DISCUSSION
We conclude that adult stem cells reside at the peripheral edge
of Descemet’s membrane in the primate eye, a region that is
commonly referred to as Schwalbe’s line. This conclusion rests
upon (1) the discovery of cells with long-term retention of
BrdU; (2) the fact that cells with BrdU retention are
immunoreactive for OCT4, a marker for stem cells; and (3)
the identification of ultrastructural characteristics for a stem
cell niche in the Schwalbe’s line region.
The cells in the Schwalbe’s line region that we identified are
very likely identical to those described by Guiseppina Raviola
in the rhesus monkey more than 30 years ago.
37
Raviola termed
the cells ‘‘Schwalbe’s line cells’’ and described them as being
arranged in a discontinuous cord of variable thickness oriented
circumferentially at the corneal periphery. Similar to our study,
the cells were reported as rich in mitochondria and to form
clusters at the tapering end of Descemet’s membrane. Based on
her additional observation of secretory granules and osmio-
philic lamellated bodies in Schwalbe’s line cells, Raviola
hypothesized that the cells produce a phospholipid material
FIGURE 2. BrdU-positive cells in the chamber angle. (A) Immunohis-
tochemical staining for BrdU (green) in cells of the TM outflow
pathway. Nuclei are stained with DAPI (blue). Arrows indicate BrdU-
positive cells in Schlemm’s canal endothelium and in the region of
Schwalbe’s line. (B,C) Quantification and statistical analysis of BrdU-
positive cells in the different quadrants of group 1 ([B], chronic BrdU)
and group 2 ([B], chronic BrdU and long-term retention) eyes. Means
6SEM are shown.
Schwalbe’s Line Stem Cells IOVS jNovember 2014 jVol. 55 jNo. 11 j7502
which is released in the aqueous humor and thus facilitates its
movement through the tissues of the sclerocorneal angle. This
hypothesis is not supported by the data of our study, since in
the two monkey eyes that were investigated by TEM, we did
not observe secretory granules and/or osmiophilic lamellated
bodies as a characteristic structural element of Schwalbe’s line
cells.
We did, however, observe cells with a high nuclear-
cytoplasmic ratio, heterochromatin-rich nuclei and a sparse
cytoplasm that were in close contact with Descemet’s
membrane and engulfed by the larger, mitochondria-rich cells.
Comparable ultrastructural characteristics have been observed
in other types of stem cells in or outside the eye.
38,39
It has
been hypothesized that adult stem cells are maintained in a
state of ‘‘stemness’’ by the presence of controlled intrinsic and
extrinsic factors in their local microenvironment, the so-called
stem cell niche.
14
Factors that are required for such a niche are
extracellular matrix components and cell-cell contacts. Based
on this concept, we hypothesize that Descemet’s membrane at
its periphery and the mitochondria-rich cell type are critical
components of the stem cell niche in Schwalbe’s line region.
In the monkey eye, Descemet’s membrane forms disconti-
nuities at its peripheral end.
29
Schwalbe’s line cells are often
seen to be in direct contact with the TM cells of its nonfiltering
anterior part through those discontinuities. It seems reasonable
to propose that this is also the route which is used after stem
cell division by resulting progenitor cells to migrate to the TM
in order to replace cells. In support of this hypothesis are
observations in monkey eyes in which TM damage leading to
cell loss was induced by long-term treatment with echothio-
phate
40
or timolol.
41
In these eyes, large clusters of elongated
cells strands were seen in the nonfiltering operculum part of
FIGURE 3. BrdU-positive cells in the trabecular meshwork outflow pathways. (A,B) Relative number of BrdU-positive cells in the different regions
of the TM outflow pathways in group 1 (A) and group 2 (B) eyes. Means 6SEM are shown. *P<0.05. **P<0.01. ***P<0.001. (C)
Immunohistochemical staining of Schlemm’s canal endothelium in a group 2 eye for BrdU (green) and CD31 (red). Cell nuclei are stained with DAPI
(blue). The arrows point toward a BrdU-/CD31-positive cell in Schlemm’s canal endothelium. (D) Immunohistochemical staining of Schlemm’s
canal endothelium in a group 2 eye for BrdU (green) and OCT4 (red). Nuclei are stained with DAPI (blue). The arrows point toward a BrdU-positive
cell in Schlemm’s canal endothelium, arrowheads mark nonnuclear labeling in the JCT.
Schwalbe’s Line Stem Cells IOVS jNovember 2014 jVol. 55 jNo. 11 j7503
the TM, which were not present at similar amounts in controls.
Overall, this observation is in agreement with the concept that
stem cell numbers expand following injury (clonogenic
capacity).
Several reports indicate that cells comparable to those
characterized in the present study in the monkey eye are
similarly localized in the Schwalbe’s line region of the human
eye. Acott and colleagues
11
studied
3
H-thymidine incorpora-
tion into trabecular cell DNA in a human corneoscleral explant
organ culture system that was treated by laser trabeculoplasty.
The authors observed a 4-fold increase in cell division and
nearly 60% of this cell division was localized to the anterior,
nonfiltering region of the trabecular meshwork where it inserts
into the cornea beneath Schwalbe’s line. In other studies using
human corneas with attached scleral rims obtained from eye
banks, BrdU-labeling and the expression of stem cell markers
like OCT4 were observed in an area just at and adjacent to the
FIGURE 4. BrdU-positive cells in Schwalbe’s line region. (A,B) Relative
number of BrdU-positive cells in Schwalbe’s line region in comparison
with that in the different regions of the TM outflow pathways in group
1(A) and group 2 (B) eyes. Means 6SEM are shown, **P<0.01. ***P<
0.001. Due to structural damage at the Schwalbe’s line, one eye could
not be included in this analysis. (C) Immunohistochemical staining of
Schwalbe’s line cells in a group 2 eye for BrdU (green) and OCT4 (red).
Nuclei are stained with DAPI (blue). Arrowheads indicate BrdU/OCT4-
positive cells in Schwalbe’s line region, while the arrow points toward
a BrdU/OCT4-negative nucleus that is stained with DAPI.
FIGURE 5. Structural characteristics of Schwalbe’s line region. (A)
Immunohistochemistry for BrdU (green) in Schwalbe’s line cells
(arrows). Nuclei are stained with DAPI (blue). (B,C) Semi-thin
meridional (B) and equatorial (C) sections through the same area as in
(A) in the eye of a different monkey. Cuboidal epithelial cells in
Schwalbe’s line form small clusters which are embedded in furrows of
Descemet’s membrane.
Schwalbe’s Line Stem Cells IOVS jNovember 2014 jVol. 55 jNo. 11 j7504
trabecular meshwork, especially when the tissue had been
wounded earlier.
42,43
Clearly, in fresh human tissues studies on
a characteristic stem cell property such as long-term BrdU
retention are difficult if impossible to perform. Moreover, in
organ culture of human tissue the expression of stem cell
molecules or the incorporation of BrdU might be under the
influence of the growth factors which are added as supplement
to the culture medium. Still, taking into account the overall
anatomical and structural similarities between the monkey and
human eye, it appears to be very likely that cells with stem cell
properties reside in region of Schwalbe’s line in the human
eye.
The two cellular populations that are in the immediate
neighborhood of Schwalbe’s line cells, TM cells and the
corneal endothelium, both take their origin from neural crest
cells that migrate to this area of the eye during develop-
ment.
44,45
It is tempting to speculate that Schwalbe’s line cells
provide a population of pluripotent stem cells that is capable
of differentiating down a TM and a corneal endothelial lineage
to replace both cell types (Fig. 7). In contrast to TM cells, SC
cells appear to be replaced by differentiated vascular
endothelial cells that are capable of mitosis. The specific
environment of SC inner wall cells that do not reside on a
complete basal lamina,
2
and are subject to continuous stretch
and strain induced by aqueous humor flow may require SC cell
regeneration when SC cells detach and become lost.
In untreated POAG, the number of TM cells is significantly
smaller than in age-matched normal eyes.
6
Another character-
istic finding in POAG is the reduction of SC cross-sectional area,
SC perimeter, and SC inner wall length.
46
It is plausible that a
cell-based therapy that leads to a repopulation of the TM
outflow pathways with differentiated TM and/or SC cells may
help to prevent or reverse the structural and functional
changes in patients that suffer from POAG.
47
The results of
the present study provide evidence for this concept and the
need for further study.
We realize that the central hypothesis of our study would be
supported considerably if, in addition to OCT4, the expression
of other molecular markers for stem cells would be shown in
Schwalbe’s line cells. While the transcription factor OCT4
alone is sufficient to reprogram human neural stem cells to
pluripotency indicating its key role as regulator of stem-
ness,
48,49
its expression has been observed in adult differen-
tiated mononuclear cells, a finding that questioned somewhat
its relevance as reliable marker for adult stem cells.
50
In
general, the expression of markers in adult stem cells depends
FIGURE 6. Ultrastructural characteristics of Schwalbe’s line cells and the putative stem cell niche. (A) Transmission electron micrographs of
Schwalbe’s line cells in the same monkey as in Figures 5B and 5C. Boxed areas in (A) are shown at higher magnification in (B) and (D). Schwalbe’s
line cells fill gaps in Descemet’s membrane and are in direct contact on their basal side with TM cells from the nonfiltrating operculum region of the
TM (A,D). Most of the cells in Schwalbe’s line region are characterized by the presence of numerous mitochondria of the tubular type (C). In
addition, smaller cells are observed with considerably less cytoplasm than the mitochondria-rich cell type (B). The smaller cells are typically
engulfed by a mitochondria-rich cell.
FIGURE 7. Semi-thin section of Schwalbe’s line cells illustrating the
concept that the cells provide a population of pluripotent stem cells
that are capable of differentiating down a TM and a corneal endothelial
lineage to replace both cell types.
Schwalbe’s Line Stem Cells IOVS jNovember 2014 jVol. 55 jNo. 11 j7505
on their specific nature and context. The cells of the trabecular
meshwork, and of the corneal stroma and endothelium
including Schwalbe’s line, all derive from the neural
crest,
44,51–53
a cell population that gives origin to an extremely
broad variety of very different tissues including but not limited
to peripheral glia and neurons, melanocytes, and cranial
mesenchyme.
54–56
Accordingly, adult neural crest-derived stem
cells, which have been isolated from various tissues such as
cornea,
57,58
iris,
59
skin,
60
palate,
61
nasal mucosa
62
or peri-
odontal ligament,
63
express a multitude of markers that are
characteristic for their broad progeny. Still, to our knowledge, a
clear-cut marker that identifies undifferentiated adult neural
crest-derived stem cells in situ has not been identified so far.
64
We are confident that the data of our study, which
characterizes in detail for the first time the specific in situ
localization of a niche for neural crest-derived stem cells in the
adult eye, will greatly facilitate the isolation of the cells
allowing their detailed molecular characterization in future
studies.
Acknowledgments
The authors thank Elke Stauber, Angelika Pach, Margit Schimmel,
Silvia Babl, and Galen Heyne for technical assistance.
Supported by grants from The Glaucoma Foundation, New York,
the Deutsche Forschungsgemeinschaft (FOR 1075, TP5), and the
Ocular Physiology Research & Education Foundation.
Disclosure: B.M. Braunger, None; B. Ademoglu, None; S.E.
Koschade, None; R. Fuchshofer, None; B.T. Gabelt, None; J.A.
Kiland, None; E.A. Hennes-Beann, None; K.G. Brunner, None;
P.L. Kaufman, None; E.R. Tamm, None
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