1. Apoptosis Gene Expression Profiling of Ex Vivo Expanded Human Limbal Epithelial Cells
Sten Raeder,1 Tor Paaske Utheim,1 Øygunn Aass Utheim,1 Savita Prabhakar,5 Yiqing Cai,2 Borghild Roald,3 Kristiane Haug,1 Anders Kvalheim,1 Edvard Berger Messelt,2 Hewen Zhang, 5
Liv Drolsum,1 Bjørn Nicolaissen,1 Torstein Lyberg,4; 1Center for Eye Research, University of Oslo, Department of Ophthalmology, Ullevål University Hospital, Norway, 2Department of
Oral Biology, Faculty of Dentistry, University of Oslo, Norway, 3Department of Pathology, Ullevål University Hospital, University of Oslo, Norway. 4Center for Clinical Research,
University of Oslo, Center for Eye research Ulleval University Kirkeveien, Oslo, 0407, Norway, 5SuperArray Bioscience Corporation, 7320 Executive Way, Suite 101, Frederick MD, 21704.
101,
Introduction
Histology and Immunostaining
Apoptosis Gene Expression Profiling using RT2Profiler™ PCR Array
Profiler™
Transplantation of ex vivo expanded human limbal epithelial cells (HLEC) is used as a therapy for limbal
therapy
stem cell deficiency (LSCD). HLEC may be cultured ex vivo by a variety of expansion protocols. Although
these protocols have shown good clinical outcomes, limbal epithelial stem cell therapy still faces challenges
epithelial
regarding surgery logistics, tissue sterility, tissue transportation, and availability of tissue. Our laboratory
transportation,
was the first to report a method for short-term organ culture (OC) eye bank storage of cultured HLEC which
shortmay be beneficial in limbal epithelial stem cell therapy.1 This study was conducted to investigate whether
conventional OC storage and Optisol-GS storage were applicable to cultured HLEC and to evaluate the
Optisolextent of cell death due to apoptosis after eye bank storage of cultured HLEC. HLEC were expanded on
intact amniotic membranes for 21 days and stored for one week at 31ºC and 23ºC in OC medium or at 5ºC in
31º
23º
5º
Optisol-GS. Cultures were fixed in formaldehyde and embedded in paraffin. Low labeling indices were
Optisolparaffin.
observed under different storage conditions as assessed by immunostaining with caspase-3 (range 0.0%immunostaining
caspase0.0%1.2%) and a DNA fragmentation assay (TUNEL, range 0.0%-2.3%). Cellular results were confirmed at the
0.0%gene expression level using the Human Apoptosis RT2Profiler™ PCR Array from SuperArray Bioscience,
Profiler™
which profiles the expression of key apoptosis genes. The results showed that pro-apoptotic genes were
results
prosignificantly down-regulated and anti-apoptotic genes were significantly up-regulated in all three storage
downantiupconditions. This study demonstrates that eye bank storage of cultured HLEC is associated with minor cell
cultured
death due to apoptosis at both the cellular level and the gene expression level. This work was supported in
expression
part by the Eastern Norway Regional Health Authority, the Norwegian Association of the Blind and Partially
Norwegian
Sighted and the Blindmission IL.
Figure 2: Organ Culture Storage of Cultured HLEC at Ambient Temperature is Superior to OC
Storage at 31°C and Optisol-GS Storage at 5°C.
31°
Optisol5°
Figure 4: Anti- and Pro-Apoptotic Genes with Greater than Three-Fold Change in Expression (p<0.05)
in Cultured Human Limbal Epithelial Cells following 1-week Storage at Three Different Temperatures
Experimental Design
Hypothesis
We hypothesized that OC storage at 31°C and Optisol-GS hypothermic storage may preserve the
characteristics of cultured HLEC. Accordingly, we compared these conventional storage methods with
the novel storage method. Furthermore, because cell death due to apoptosis has been reported in
human corneal epithelium after OC culture storage2 and hypothermic storage3, we studied expression
of apoptosis-regulatory genes and examined apoptosis markers in cultured HLEC following eye bank
storage.
Functional Gene
Grouping
TNF Ligand Family
Gene Symbol
1-week organ culture
storage at 31ºC
Fold-Change
1-week organ culture
storage at 23ºC
Fold-Change
Bcl-2 Family
14.25
TNF
TNF Receptor Family
FASLG
-18.24
TNFSF10
1-week Optisol-GS
storage at 5ºC
Fold-Change
-4.21
TNFRSF9
-4.09
BAG4
BAK1
-14.3
-5.07
7.65
-7.44
-7.5
BCL2
18.87
BCL2A1
-6.13
BCL2L11
BNIP2
-4.98
8.72
4.7
11.61
11.58
BNIP3L
13.27
HRK
-3.01
-3.13
-32.41
-22.96
-27.47
-16.64
MCL1
IAP Family
-8.96
BIRC1
BIRC6
HLEC cultures were fixed in neutral-buffered 4% formaldehyde and embedded in paraffin. Serial sections of 5
µm were stained with haematoxylin and eosin (H&E). Immunohistochemistry was performed with a panel of
antibodies for markers of human ocular surface epithelia. Histological evaluation and semiquantitative
immunohistochemical localization of the epithelial markers were carried out by two independent investigators
using a microscope at a magnification of 400X. Results: The ultrastructure was preserved at 23ºC, while storage
at 31ºC and 5ºC was associated with enlarged intercellular spaces, separation of desmosomes, and detachment
of epithelial cells. Cultured HLEC remained undifferentiated under all storage conditions.
BIRC8
CARD Family
7.58
5.2
-6.69
APAF1
4.48
CARD6
6.14
3.42
CARD8
30.16
10.36
CASP5
17.81
11.53
CASP9
-4.05
PYCARD
-5.67
RIPK2
Death Domain Family
9.44
4.93
TRADD
CIDE Domain Family
16.72
FAS
Figure 3: Quantification of Apoptotic Cells
FADD
-9.11
-4.25
p53 & DNA Damage
23.86
11.75
15.21
17.79
-12.23
-8.85
CIDEB
10.21
ABL1
10.46
CASP6
A
15.78
GADD45A
Culture Conditions
3-week HLEC (n=48) cultures were either organ-cultured at 31°C (n=12) or 23°C (n=12) or stored in
Optisol-GS at 5°C (n=12) in a closed container for one week. Figure 1 explains in brief as to how the
human limbal tissue from human donor eyes were cultured on intact amniotic membranes and stored
in the eye bank.
10.8
Figure 4. RNA was isolated from the formalin-fixed paraffin-embedded (FFPE) tissue using SuperArray’s
)
ArrayGradeTM FFPE RNA Isolation Kit (GA-023). RNA (40 ng) was amplified and reverse transcribed using a
modified version of the TrueLabeling PicoAMP Kit (GA-130) and the RT2 PCR Array First Strand Kit (C-02)
from SuperArray Bioscience. The RT2Profiler™ Human Apoptosis PCR Array (APHS-012) from SuperArray
Bioscience was used to analyze the mRNA levels of 84 key genes involved in apoptosis, in a 384-well format.
Three biological replicates were included from each experimental group. Relative changes in gene expression
were calculated using the ΔΔCt method. Results: Following storage at 23ºC and 5ºC, down-regulation of
BCL2A1 and BIRC1, and reduced expression of TNF receptor signaling components (TNF and TRADD) was
revealed suggesting a reduction in nuclear factor-κB activity. Under all storage conditions, expression of BNIP2
was up-regulated, whereas MCL1 expression was down-regulated.
FIGURE 1. Eye Bank Storage of Cultured Human Limbal Epithelial Cells (HLEC)
B
Apoptotic and labeling index (%)
4
3,5
3
H&E apoptotic index
Caspase-3 labeling index
TUNEL labeling index
Conclusions
2,5
2
1,5
1
0,5
0
3-week HLEC
cultures at 37ºC
1-week OC storage 1-week OC storage
at 31ºC
at 23ºC
1-week Optisol-GS
storage at 5ºC
Figure 3A demonstrates H&E staining (top), cleaved caspase-3 immunohistochemistry (middle), and TUNEL
staining (bottom) of cultured human limbal epithelial cells after one week’s organ culture storage at 23ºC. H&E
staining demonstrates an apoptotic epithelial cell with circular nuclear fragments (arrow). Cleaved caspase-3positive surface cells have cytoplasmic immunoreactivity and well defined nuclear membranes (arrowheads). A
TUNEL positive surface cell (arrowhead) is also observed. (Original magnification: 400X) Figure 3B contains a
histogram demonstrating the H&E apoptotic index, caspase-3 labeling index, and TUNEL labeling index in
cultured human limbal epithelial cells after three weeks’ culture and one week’s storage at the three different
temperatures. Results are expressed as mean percent of the apoptotic or labeling index in the individual
experimental groups. Error bars denote 1 SE. Results: No significant increase in cleaved caspase-3 or TUNEL
:
staining was observed in response to eye bank storage under any of the three storage conditions.
Our results indicate that OC storage of cultured HLEC at ambient temperature is superior to OC
storage at 31°C and Optisol-GS storage at 5°C as the original layered structure of cultured HLEC
is preserved at 23°C storage.
Apoptosis is minimal following eye bank storage of cultured HLEC, though multi-gene profiling
revealed interesting alterations in gene expression in cultured HLEC.
Eye bank storage of cultured HLEC may provide a reliable source of tissue for treating limbal stem
cell deficiency, although its feasibility for clinical use has to be evaluated further.
References
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preserving
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in organInvestigative Ophthalmology & Visual Science. 2001;42:1757-1761.
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