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Ms. Deeksha K Reg no: 165/Jan 2015 Junior research fellow Yenepoya University

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 :

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Ms. Deeksha K Reg no: 165/Jan 2015 Junior research fellow Yenepoya University

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  1. Synopsis presentation Oxidative damage to limbal stem cells of eye in response to bright and ultraviolet light and its associated mechanisms Ms. Deeksha KReg no: 165/Jan 2015Junior research fellowYenepoya University Research Guide: Dr. Cynthia Arunachalam (Lt Col) Dept. of Ophthalmology Yenepoya University Research Co-Guide: Dr. Bipasha Bose Yenepoya Research Centre Yenepoya University

  2. Contents • 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

  3. 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

  4. Introduction Contd. • Limbal epithelial and limbalstromal 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

  5. 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)

  6. 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

  7. Introduction Contd. Oxidative stress and signaling pathways •OH, H2O2,•O−2 Dayem et al., 2010

  8. Literaturesurvey

  9. Literaturesurvey

  10. Literaturesurvey

  11. Socialrelevance • 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.

  12. Aim Aim To investigate the cellular and molecular changes in limbal epithelial stem cells (LESCs) and limbalstromal stem cells (LSSCs) of the eye in response to bright and ultraviolet light and their associated mechanisms Objectives • Isolation of LESCs and LSSCs from the eyes of mouse and human. • Characterization and expansion of LESCs and LSSCs. • Exposure of LESCs and LSSCs to bright light and ultraviolet light and assessment of cellular and molecular changes and its associated mechanisms. • Hypothesis UV and bright light will cause oxidative damage to the Limbal stem cells of the eye leading to LSCD Objectives Hypothesis

  13. Methodology • Subjects under study • Mice • Human

  14. 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)

  15. 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

  16. Objective-1 Methodology contd. Isolation of limbal epithelial and stromal stem cells From mice From human Mouse will be anaesthetized Limbal tissues (2x2mm width) will be dissected during cataract surgery Dissection along the limbus (2-3mm width) 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)

  17. 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

  18. 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/ cytoplasmicratio Measurement of Reactive oxygen species (ROS) production- DCFDA method Measurement of antioxidant enzymes- Antioxidant enzyme assay kits (SOD, CAT,GPX etc)

  19. 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

  20. Research/studyplan

  21. 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).

  22. Timeline of the project

  23. Budget plan

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  25. References • Chen, S.Y. and Xie, H.T. (2012). Isolation and Expansion of Human LimbalStromal Niche Cells. Investigative Ophthalmology & Visual Science, 53(14), pp.3499-3499. • Dayem, A.A., Choi, H.Y., Kim, J.H. and Cho, S.G. (2010). Role of oxidative stress in stem, cancer, and cancer stem cells. Cancers, 2(2), pp.859-884. • Demicco, E.G., Kavanagh, K.T., Romieu-Mourez, R., Wang, X., Shin, S.R., Landesman-Bollag, E., Seldin, D.C. and Sonenshein, G.E. (2005). RelB/p52 NF-κB complexes rescue an early delay in mammary gland development in transgenic mice with targeted superrepressorIκB-α expression and promote carcinogenesis of the mammary gland. Molecular and cellular biology, 25(22), pp.10136-10147. • Denk, A., Wirth, T. and Baumann, B. (2000). NF-κB transcription factors: critical regulators of hematopoiesis and neuronal survival. Cytokine & growth factor reviews, 11(4), pp.303-320. • Dua, H.S. and Azuara-Blanco, A. (2000). Limbal stem cells of the corneal epithelium. Survey of ophthalmology, 44(5), pp.415-425. • Dua, H.S., Joseph, A., Shanmuganathan, V.A. and Jones, R.E. (2003). Stem cell differentiation and the effects of deficiency. Eye, 17(8), pp.877-885.

  26. References • Dua, H.S., Shanmuganathan, V.A., Powell-Richards, A.O., Tighe, P.J. and Joseph, A. (2005). Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche. British Journal of Ophthalmology, 89(5), pp.529-532. • Ghosh, S., May, M.J. and Kopp, E.B. (1998). NF-κB and Rel proteins: evolutionarily conserved mediators of immune responses. Annual review of immunology, 16(1), pp.225-260. • Hui, H., Hu, M., Zhao, X. and Tang, Y. (2011). Stem Cells: General Features and Characteristics.Intech Open Acess Publisher • Kang, H.B., Kim, Y.E., Kwon, H.J., Sok, D.E. and Lee, Y. (2007). Enhancement of NF-κ B Expression and Activity Upon Differentiation of Human Embryonic Stem Cell Line SNUhES3. Stem cells and development, 16(4), pp.615-624. • Katikireddy, K.R. and Jurkunas, U.V. (2016). LimbalStromal Tissue Specific Stem Cells and Their Differentiation Potential to Corneal Epithelial Cells.Methods in molecular biology (Clifton, NJ), 1341, p.437. • Kennedy, N.J., Cellurale, C. and Davis, R.J. (2007). A radical role for p38 MAPK in tumor initiation. Cancer cell, 11(2), pp.101-103. • Kim, M.K., Lee, J.L., Shin, K.S., Jung, G.A., Wee, W.R., Lee, J.H., Park, K.S. and Son, Y.S. (2006). Isolation of putative corneal epithelial stem cells from cultured limbal tissue. Korean Journal of Ophthalmology, 20(1), pp.55-61.

  27. References • Klenkler, B. and Sheardown, H. (2004). Growth factors in the anterior segment: role in tissue maintenance, wound healing and ocular pathology.Experimental eye research, 79(5), pp.677-688. • Ksander, B.R., Kolovou, P.E., Wilson, B.J., Saab, K.R., Guo, Q., Ma, J., McGuire, S.P., Gregory, M.S., Vincent, W.J., Perez, V.L. and Cruz-Guilloty, F. (2014). ABCB5 is a limbal stem cell gene required for corneal development and repair. Nature. • Liou, H.C. and Baltimore, D. (1993). Regulation of the NF-ηB/rel transcription factor and IηB inhibitor system. Current opinion in cell biology, 5(3), pp.477-487. • Longstreth, J., De Gruijl, F.R., Kripke, M.L., Abseck, S., Arnold, F., Slaper, H.I., Velders, G., Takizawa, Y. and Van derLeun, J.C. (1998). Health risks.Journal of Photochemistry and Photobiology B: Biology, 46(1), pp.20-39. • López-Paniagua, M., Nieto-Miguel, T., de la Mata, A., Dziasko, M., Galindo, S., Rey, E., Herreras, J.M., Corrales, R.M., Daniels, J.T. and Calonge, M. (2016). Comparison of functional limbal epithelial stem cell isolation methods.Experimental eye research, 146, pp.83-94. • Marchitti, S.A., Chen, Y., Thompson, D.C. and Vasiliou, V. (2011). Ultraviolet radiation: cellular antioxidant response and the role of ocular aldehydedehydrogenase enzymes. Eye & contact lens, 37(4), p.206.

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  29. References • Notara, M., Refaian, N., Braun, G., Steven, P., Bock, F. and Cursiefen, C. (2015). Short-term uvb-irradiation leads to putative limbal stem cell damage and niche cell-mediated upregulation of macrophage recruiting cytokines.Stem cell research, 15(3), pp.643-654. • Polisetti, N., Zenkel, M., Menzel‐Severing, J., Kruse, F.E. and Schlötzer‐Schrehardt, U. (2016). Cell Adhesion Molecules and Stem Cell‐Niche‐Interactions in the Limbal Stem Cell Niche. STEM CELLS, 34(1), pp.203-219. • Priya, C.G., Arpitha, P., Vaishali, S., Prajna, N.V., Usha, K., Sheetal, K. and Muthukkaruppan, V. (2011). Adult human buccal epithelial stem cells: identification, ex-vivo expansion, and transplantation for corneal surface reconstruction. Eye, 25(12), pp.1641-1649. • Qiu, P., Pan, P.C. and Govind, S. (1998). A role for the Drosophila Toll/Cactus pathway in larval hematopoiesis. Development, 125(10), pp.1909-1920. • Saito, H., Yokoyama, H. and Hibi, T. (2006). Superoxide anion and reactive oxygen species and redox regulation in the liver. The Japanese journal of gastro-enterology, 103(1), pp.15-22. • Schlötzer-Schrehardt, U. and Kruse, F.E. (2005). Identification and characterization of limbal stem cells. Experimental eye research, 81(3), pp.247-264.

  30. References • Shortt, A.J., Secker, G.A., Munro, P.M., Khaw, P.T., Tuft, S.J. and Daniels, J.T. (2007). Characterization of the limbal epithelial stem cell niche: novel imaging techniques permit in vivo observation and targeted biopsy of limbal epithelial stem cells. Stem cells, 25(6), pp.1402-1409. • Takács, L., Tóth, E., Losonczy, G., Szanto, A., Bähr-Ivacevic, T., Benes, V., Berta, A. and Vereb, G. (2011). Differentially expressed genes associated with human limbal epithelial phenotypes: new molecules that potentially facilitate selection of stem cell-enriched populations. Investigative ophthalmology & visual science, 52(3), pp.1252-1260. • Trosan, P., Svobodova, E., Chudickova, M., Krulova, M., Zajicova, A. and Holan, V. (2012). The key role of insulin-like growth factor I in limbal stem cell differentiation and the corneal wound-healing process. Stem cells and development, 21(18), pp.3341-3350. • Vimalin, J., Gupta, N., Jambulingam, M., Padmanabhan, P. and Madhavan, H.N. (2012). The Effect of Riboflavin–UV-A Treatment on Corneal Limbal Epithelial Cells—A Study on Human Cadaver Eyes. Cornea, 31(9), pp.1052-1059. • Weisiger, R.A. and Fridovich, I. (1973). Mitochondrial superoxide dismutase site of synthesis and intramitochondrial localization. Journal of Biological Chemistry, 248(13), pp.4793-4796.

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