Synopsis presentation
Oxidative damage to limbal stem cells of eye in
response to bright and ultraviolet light and its
associated mechanisms
Ms. Deeksha K
Reg no: 165/Jan 2015
Junior research fellow
Yenepoya University
Research Guide:
Dr. Cynthia Arunachalam (Lt Col)
Dept. of Ophthalmology
Yenepoya University
Research Co-Guide:
Dr. Bipasha Bose
Yenepoya Research Centre
Yenepoya University
1
Contents
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Introduction and background
Literature survey
Social relevance of the study
Aim, objectives, hypothesis
Methodology
Procedures for data collection
Research/study plan
Statistical analysis
Timeline of the project
Budget plan for the project
References
2
Introduction
• Limbus – region where the corneal stem cells/limbal stem
cells reside (Dua et al., 2000)
• Identified the stem cell population first time in the Palisades
of Vogt of limbus (Pellegrini et al. 1999)
• It acts as a barrier between cornea and conjunctiva (Dua et
al., 2000)
www.eophtha.com
3
Introduction
Contd.
• Limbal epithelial and limbal stromal are the two kinds of limbal
stem cells for corneal epithelial regeneration.
• LSSC restore corneal transparency (Du et al.; 2009)
 Limbal Stem Cell Deficiency(LSCD)
• Failure in the selfrenewing and barrier function of the limbus
stem cells
• Failure in corneal epithelial healing
• Phenotypic difference in conjunctival epithelium
• Associated blood vessels invade corneal surface
• Chronic ocular discomfort and pain.
• Corneal conjunctivalization leads to- loss
of corneal clarity and
vision impairment (Dua et al., 2000)
www.healio.com
LSCD eye
4
Introduction
 Prevalence of LSCD in India:
– India needs 1 Lakh corneal transplants a year. Of these
about 6000 need limbal stem cell treatment a year
(Jotwani , 2013)
5
Introduction
Contd.
Causes of LSCD:
• Genetic causes – Aniridia , epidermal dysplasia (Ramaesh et al.;2005)
•Acquired causes
•Chemical and thermal injury to the front of the eye.
•Antimetabolites
•Ocular surgery involving limbus
•Microbial infection extending to limbus
•Contact lens associated (Clinch et al.;1992) (Sejpal et al., 2013)
No reports regarding the UV or bright light induced oxidative
damage to the limbal stem cells of the eye
6
Introduction
Contd.
Oxidative stress and signaling pathways
Oxidative stress
Plasma membrane
Bright
light
Infrared
rays
UV rays
ROS
•OH,
Iк-Bα
Bcl-1, Bcl-xL, TRAF-1/2
H2O2,•O−2
MEK-1
NF-кB
NF-кB
Cytoplasm
P38, JNK, ERK1/2
Nucleus
Dayem et al., 2010
C-JUN/AP-1
Cyclin D1, p21, p63
7
Literature survey
Sr.
No
Study /Year (Reference)
Findings
1
Pellegrini et al., 1999
Identified limbal stem cells in interpalisades of vogt
2
AJ et al., 2007
Limbus acts as physical barrier between
cornea and conjunctiva
3
Dua et al., 2003
Corneal epithelial homeostasis and
regeneration
4
Dua et al., 2010
Limbal stem cells have role in corneal
epithelial healing
5
Dua et al., 2010
LSCD is the major cause of blindness
worldwide
6
Ursula et al., 2005
Identified ABCG2 as limbal stem cell
marker
7
Ksander et al., 2014
ABCB5 is a limbal stem cell gene required
for corneal development and repair
8
Literature survey
Sr.
No
Study /Year (Reference)
Findings
8
Ahmad et al., 2010
LSCD is the condition stem cell function
and barrier function of the limbus is lost
9
Arpitha et al., 2011
Limbal epithelium has high N/c ratio,
express high levels of nuclear protein p63
10
Podskochy & Fagerholm 1998;
Podskochy et al., 2000
UVB causes apoptosis and loss of
epithelial cells without causing any deep
damage to underlying corneal layers
11
Rogers et al., 2004
UV-induced corneal injury result from
oxidative stress by the localized
generation of reactive oxygen species
(ROS), including superoxide anion,
hydrogen peroxide, and hydroxyl radical
by corneal epithelial cells
12
Dayem et al., 2010
Reactive oxygen species are considered
as the most significant mutagens in stem
cells
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Literature survey
Sr.
No
Study /Year (Reference)
Findings
13
Norman et al., 2007
ROS can stimulate cellular proliferation
and activate survival pathways via several
signalling mechanisms
14
Notara et al., 2015
Short term UVB radiation leads to
putative limbal stem cell damage and
niche cell mediated upregulation of
macrophage recruiting cytokines
15
Katikireddy et al., 2016
Limbal stromal stem cells differentiate
into corneal epithelial cells
16
Lopez et al., 2016
Cell suspension method is the best
method to isolate LESCs
17
Polisetti et al., 2016
Identified novel cell adhesion molecules
and their stem cell niche interactions in
the limbal stem cell niche
10
Social relevance
• Developing cell therapies for the treatment of corneal LSCD
is a priority area in the field of regenerative medicine.
• The study attempts to discern the molecular mechanisms
responsible for limbal stem cell depletion in response to UV
and bright light.
• Findings of the study are anticipated to give clues to
modulate the signaling pathways to overcome LSCD that
can be used as a therapy.
11
Aim
Aim
To investigate the cellular and molecular changes in limbal
epithelial stem cells (LESCs) and limbal stromal stem cells
(LSSCs) of the eye in response to bright and ultraviolet light
and their associated mechanisms
Objectives
Objectives
1. Isolation of LESCs and LSSCs from the eyes of mouse and
human.
2. Characterization and expansion of LESCs and LSSCs.
3. Exposure of LESCs and LSSCs to bright light and ultraviolet light
and assessment of cellular and molecular changes and its
associated mechanisms.
• Hypothesis
Hypothesis
UV and bright light will cause oxidative damage to the Limbal
stem cells of the eye leading to LSCD
12
Methodology
 Subjects under study
– Mice
– Human
13
Procedures for data collection
• Mice
–
–
–
–
–
C57BL6J and inbred swiss albino
male
5 week old mice
weight approximately 23-25g
Sample size - 24 mice (Holan et al., 2010)
14
Procedures for data collection
Contd.
 Human subject selection criteria
• Inclusion criteria
– Patient undergoing cataract surgery
– Age group of above 20 years
– Sample size- 50 patients (Chen et al., 2012)
• Exclusion criteria
– Patient with any ocular surface disease
– Patient who has undergone any pterygium surgery
– Patient who has undergone any ocular surgery in the past
– Patient with any connective tissue/immunological systemic
disorders
– Patient on any ocular topical medication
– Patient with any history of any ocular trauma, mechanical/ chemical
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Objective-1
Methodology contd.
Isolation of limbal epithelial and stromal stem cells
From mice
From human
Mouse will be anaesthetized
Dissection along the
limbus (2-3mm width)
Limbal tissues (2x2mm width) will
be dissected during cataract
surgery
PBS washes with antibiotics (2-3 times)
Enzymatic digestion (Trypsin – EDTA / Dispase)
Cells will be cultured in KSFM /DMEM media with growth factors
Incubated at 5% CO2
Cell viability test
(Holan et al., 2010; Kim et al., 2007; Chen et al., 2012)
16
Objective-2
Methodology contd.
Characterization and expansion of limbal epithelial and
stromal stem cells
Primary cultured mouse and human limbal epithelial and stromal cells
Fluorescence activated cell sorting of LESCs and LSSCs for ABCG2
Expansion of pure population of ABCG2 positive cells
Gene expression analysis
Western blot- ABCG2, p63
RT-PCR- Limbal stem cell markers, corneal differentiation markers,
conjunctival markers
Immunostaining- Limbal stem cell markers, corneal differentiation
markers, conjunctival markers
Experiments will be done using standardized protocols
17
Objective-3
Methodology contd.
Exposure of LESCs and LSSCs to bright light and ultraviolet
light and assessment of cellular and molecular changes
and its associated mechanisms
ABCG2 positive limbal epithelial and stromal cells
Treatment of cells with UV radiation -UVA(400-320nm), UVB (320 nm - 290 nm) and
UVC (254nm) of different energies and bright light (10,000 lux)
Controls along with triplicates will be maintained
Morphological assessment – Nuclear/ cytoplasmic ratio
Measurement of Reactive oxygen species (ROS) production- DCFDA method
Measurement of antioxidant enzymes- Antioxidant enzyme assay kits
(SOD, CAT,GPX etc)
18
Objective-3
Methodology contd.
qRT-PCR and immunostaining- limbal stem cell and oxidative marker genes
(8-OHdG, MDA, HNE)
Assessment of cell signaling mechanisms associated with oxidative stress
(MAPK,ERK, JNK, NFKB pathway)
Gene expression analysis
qRT-PCR, western blots, flow cytometry, immunostaining and ELISA
Experiments will be done using standardized protocols
19
Research/study plan
Isolation of limbal
epithelial and stromal
stem cells from
mouse/ human
RT-PCR for limbal stem
cell markers, corneal
differentiation markers,
conjunctival markers
Detection of reactive
oxygen species by 2’,
7’ dichlorofluorescein
diacetate
(DCFDA)
method
FACS sorting of limbal
epithelial and stromal
stem cells for ABCG2
marker
Morphological
assessment of limbal
stem cells
Immunostaining for
limbal markers and
oxidative markers
Expansion of pure
ABCG2 positive cells
Treatment of limbal
stem cells with UVA,
UVB and UVC
radiations of different
energies
Assessment of cell
signalling mechanisms
associated with
oxidative stress
(MAPK, ERK, JNK, NFKB
pathway)
20
Statistical analysis
• Data will be expressed in mean ± standard deviation.
• Student’s t test will be done for comparing different groups.
• p<0.05 will be considered to be statistically significant.
• Data will be analyzed using SPSS software (version 22).
21
Timeline of the project
22
Budget plan
Sl.
no
Item
1
Price (INR)
Ist year
2nd year
Total
Cell culture reagents: media, antibiotics,
serum, trypsin, dispase, growth factors,
cryopreservation media
75,000
50,000
1,25,000
2
Culture flasks, culture plates, disposable
pipettes, pipette tips, matrigel, syringe
filters, 50ml and 15ml centrifuge tubes,
eppendorf tubes, etc
70,000
--------------
70,000
3
Mice
5,000
5,000
10,000
4
Primary and secondary antibodies for
FACS, immunofluorescence etc; primers for
PCR, western blot reagents
1,50,000
1,50,000
3,00,000
5
Antioxidant enzyme assay kits, reactive
oxygen species detection dye
25,000
--------------
25000
6
Several minor chemicals and reagents
30,000
20,000
50,000
Grand Total
3,55,000
2,25,000
5,80,000
23
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