Substrate engineering using naturally biomimicking corneal cell topography for preserving stemness of corneal limbal epithelial-stem cells

Document Type : Original Article

Authors

1 Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran

2 Department of Tissue Engineering & Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran

3 Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran

4 Department of Hematology and Blood Banking, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran

5 Basir Eye Health Research Center, Iran University of Medical Sciences, Tehran, Iran

6 Eye Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran

10.22038/ijbms.2025.86110.18601

Abstract

Objective(s): Substrate engineering is one of the attractive fields of changing cell behavior and fate, especially for stem cell (SC) therapies. The SC pool is an essential factor in transplantation outcomes. Here, the objective was to preserve the stemness of the cornea’s limbal epithelial stem cell (LESC) using naturally biomimicking corneal cell topography.
Materials and Methods: A cell-imprinted substrate was prepared using the natural topography of rabbit cornea’s LESC. The LESC cells were characterized by immunostaining (ABCG2 and Cytokeratin-12), then re-cultivated on a topography mold (imprinted PDMS), on FLAT PDMS (without any pattern), and the control group (tissue culture plate). Ultimately, an alkaline burn model was created on a rabbit’s cornea, and the effectiveness of cell-imprinted molds as implants for healing corneal wounds was examined in vivo.  
Results: The in vitro results showed that imprinted PDMS kept LESC cells in a state of stemness with high expression of ∆NP63 and ABCG2 genes (stemness-associated genes) compared to the other two groups and low Cytokeratin-3 and -12 expression (as differentiation-related genes). In vivo studies showed a more significant number of cells and the expression of the ABCG2 gene in the imprinted PDMS group. In contrast, higher expressions of the ∆Np63 gene and more stratification were observed in the control group (no treatment). Histological studies showed that the imprinted PDMS group had normal morphology with fully organized collagens. 
Conclusion: The results of LESC cultured on imprinted PDMS suggested that LESC cell imprinting could be an excellent substrate for LESC expansion and preserve their stemness for cell therapy.

Keywords

Main Subjects

References

1. Manoochehrabadi T, Solouki A, Majidi J, Khosravimelal S, Lotfi E, Lin K, et al. Silk biomaterials for corneal tissue engineering: From research approaches to therapeutic potentials; A review. Int J Biol Macromol 2025; 305: 1-24.
2. Singh V, Tiwari A, Kethiri AR, Sangwan VS. Current perspectives of limbal-derived stem cells and its application in ocular surface regeneration and limbal stem cell transplantation. Stem Cells Transl Med 2021; 10: 1121-1128. 
3. Shukla S, Shanbhag SS, Tavakkoli F, Varma S, Singh V, Basu S. Limbal epithelial and mesenchymal stem cell therapy for corneal regeneration. Curr Eye Res 2020; 45: 265-277.
4. Yanez-Soto B, Liliensiek SJ, Gasiorowski JZ, Murphy CJ, Nealey PF. The influence of substrate topography on the migration of corneal epithelial wound borders. Biomaterials. 2013; 34: 9244-9251.
5. Gouveia RM, Lepert G, Gupta S, Mohan RR, Paterson C, Connon CJ. Assessment of corneal substrate biomechanics and its effect on epithelial stem cell maintenance and differentiation. Nat Commun 2019; 10: 1-17.
6. Xiong S, Gao H, Qin L, Jia YG, Ren L. Engineering topography: Effects on corneal cell behavior and integration into corneal tissue engineering. Bioact Mater 2019; 4: 293-302.
7. Kang KB, Lawrence BD, Gao XR, Guaiquil VH, Liu A, Rosenblatt MI. The effect of micro-and nanoscale surface topographies on silk on human corneal limbal epithelial cell differentiation. Sci Rep 2019; 9: 1-8. 
8. Haramshahi SMA, Bonakdar S, Moghtadaei M, Kamguyan K, Thormann E, Tanbakooei S, et al. Tenocyte-imprinted substrate: A topography-based inducer for tenogenic differentiation in adipose tissue-derived mesenchymal stem cells. Biomed Mater 2020; 15: 1-7. 
9. Majidi M, Pakzad S, Salimi M, Azadbakht A, Hajighasemlou S, Amoupour M, et al. Macrophage cell morphology‐imprinted substrates can modulate mesenchymal stem cell behaviors and macrophage M1/M2 polarization for wound healing applications. Biotechnol Bioeng 2023; 120: 3638-3654.
10. Nikkhah M, Edalat F, Manoucheri S, Khademhosseini A. Engineering microscale topographies to control the cell–substrate interface. Biomaterials 2012; 33: 5230-5246.
11. Vattulainen M, Ilmarinen T, Viheriälä T, Jokinen V, Skottman H. Corneal epithelial differentiation of human pluripotent stem cells generates ABCB5+ and Np63α+ cells with limbal cell characteristics and high wound healing capacity. Stem Cell Res Ther 2021; 12: 1-17.
12. Morita M, Fujita N, Takahashi A, Nam ER, Yui S, Chung CS, et al. Evaluation of ABCG 2 and p63 expression in canine cornea and cultivated corneal epithelial cells. Vet Ophthalmol 2015; 18: 59-68.
13. Baba K, Sasaki K, Morita M, Tanaka T, Teranishi Y, Ogasawara T, et al. Cell jamming, stratification and p63 expression in cultivated human corneal epithelial cell sheets. Sci Rep 2020; 10: 1-8.
14. Shi L, Stachon T, Käsmann-Kellner B, Seitz B, Szentmáry N, Latta L. Keratin 12 mRNA expression could serve as an early corneal marker for limbal explant cultures. Cytotechnology 2020; 72: 239-245.
15. Kao WW-Y. Keratin expression by corneal and limbal stem cells during development. Exp Eye Res 2020; 200: 1-8.
16. Kethiri AR, Basu S, Shukla S, Sangwan VS, Singh V. Optimizing the role of limbal explant size and source in determining the outcomes of limbal transplantation: An in vitro study. PLoS One 2017; 12: 1-17.
17. Utheim TP, Lyberg T, Ræder S. The Culture of Limbal Epithelial Cells. In: Wright B, Connon CJ, editors. Corneal Regenerative Medicine: Methods and Protocols. Totowa, NJ: Humana Press; 2013. p. 103-29.
18. Zhou J, Khodakov DA, Ellis AV, Voelcker NH. Surface modification for PDMS‐based microfluidic devices. Electrophoresis 2012; 33: 89-104.
19. Alizadeh S, Samadikuchaksaraei A, Jafari D, Orive G, Dolatshahi‐Pirouz A, Pezeshki‐Modaress M, et al. Enhancing diabetic wound healing through improved angiogenesis: The role of emulsion‐based core‐shell micro/nanofibrous scaffold with sustained CuO nanoparticle delivery. Small 2024; 20: 1-18.
20. Gouveia RM, Vajda F, Wibowo JA, Figueiredo F, Connon CJ. YAP, ΔNp63, and β-catenin signaling pathways are involved in the modulation of corneal epithelial stem cell phenotype induced by substrate stiffness. Cells 2019; 8: 1-10. 
21. Lotfi E, Kholghi A, Golab F, Mohammadi A, Barati M. Circulating miRNAs and lncRNAs serve as biomarkers for early colorectal cancer diagnosis. Pathol Res Pract 2024; 255: 1-6.
22. Liu L, He H, Liu J. Advances on non-genetic cell membrane engineering for biomedical applications. Polymers 2019; 11: 1-26.
23. Fernandez-Buenaga R, Aiello F, Zaher SS, Grixti A, Ahmad S. Twenty years of limbal epithelial therapy: An update on managing limbal stem cell deficiency. BMJ Open Ophthalmol 2018; 3: 1-7.
24. Rahvar M, Lakalayeh GA, Nazeri N, Karimi R, Borzouei H, Ghanbari H. Micro/nanoscale surface engineering to enhance hemocompatibility and reduce bacterial adhesion for cardiovascular implants. Mater Chem Phys 2022; 289: 1-14.
25. Rahvar M, Ahmadi Lakalayeh G, Nazeri N, Marouf BT, Shirzad M, Najafi T. Shabankareh A, et al. Assessment of structural, biological and drug release properties of electro-sprayed poly lactic acid-dexamethasone coating for biomedical applications. Biomed Eng Lett 2021; 11: 393-406.
26. Rahvar M, Manoochehrabadi T, Ahmadi Lakalayeh G, Barzegar Z, Karimi R, Ghanbari H. Development of a highly hydrophobic micro/nanostructured nanocomposite coating of PLA-PEG-cloisite 20A nanoclay with excellent hemocompatibility and rapid endothelialization properties for cardiovascular applications. ACS Appl Mater Interfaces 2025; 17: 4579-45.
27. Shen L, Zhu J. Heterogeneous surfaces to repel proteins. Adv Colloid Interface Sci 2016; 228: 40-54.
28. Huang Q, Lin L, Yang Y, Hu R, Vogler EA, Lin C. Role of trapped air in the formation of cell-and-protein micropatterns on superhydrophobic/superhydrophilic microtemplated surfaces. Biomaterials 2012; 33: 8213-8220.
29. Cha KJ, Park KS, Kang SW, Cha BH, Lee BK, Han IB, et al. Effect of replicated polymeric substrate with lotus surface structure on adipose‐derived stem cell behaviors. Macromol Biosci 2011; 11: 1357-1363.
30. Mahmoudi M, Bonakdar S, Shokrgozar MA, Aghaverdi H, Hartmann R, Pick A, et al. Cell-imprinted substrates direct the fate of stem cells. ACS Nano 2013; 7: 8379-8384.
31. Gao J, Raghunathan VK, Reid B, Wei D, Diaz RC, Russell P, et al. Biomimetic stochastic topography and electric fields synergistically enhance directional migration of corneal epithelial cells in a MMP-3-dependent manner. Acta Biomater 2015; 12: 102-12.
32. Raghunathan VK, Dreier B, Morgan JT, Tuyen BC, Rose BW, Reilly CM, et al. Involvement of YAP, TAZ and HSP90 in contact guidance and intercellular junction formation in corneal epithelial cells. PloS One 2014; 9: 1-14.
33. Song W, Mano JF. Interactions between cells or proteins and surfaces exhibiting extreme wettabilities. Soft Matter 2013; 9: 2985-2999.
34. Zhang W, Chen J, Backman LJ, Malm AD, Danielson P. Surface topography and mechanical strain promote keratocyte phenotype and extracellular matrix formation in a biomimetic 3D corneal model. Adv Healthc Mater 2017; 6: 1-11. 
35. Raghunathan V, McKee C, Cheung W, Naik R, Nealey PF, Russell P, et al. Influence of extracellular matrix proteins and substratum topography on corneal epithelial cell alignment and migration. Tissue Eng part A 2013; 19: 1713-1722.
36. McHugh KJ, Saint‐Geniez M, Tao SL. Topographical control of ocular cell types for tissue engineering. J Biomed Mater Res B Appl Biomater 2013; 101: 1571-1584.
37. Diehl K, Foley J, Nealey P, Murphy C. Nanoscale topography modulates corneal epithelial cell migration. J Biomed Mater Res A 2005; 75: 603-611.
38. Kang KB, Lawrence BD, Gao XR, Luo Y, Zhou Q, Liu A, et al. Micro-and nanoscale topographies on silk regulate gene expression of human corneal epithelial cells. Invest Ophthalmol Vis Sci 2017; 58: 6388-6398.
39. Tocce E, Liliensiek S, Broderick A, Jiang Y, Murphy K, Murphy C, et al. The influence of biomimetic topographical features and the extracellular matrix peptide RGD on human corneal epithelial contact guidance. Acta Biomater 2013; 9: 5040-5051.
40. Lawrence BD, Pan Z, Rosenblatt MI. Silk film topography directs collective epithelial cell migration. PloS One. 2012; 7: 1-12. 
41. Lawrence BD, Pan Z, Liu A, Kaplan DL, Rosenblatt MI. Human corneal limbal epithelial cell response to varying silk film geometric topography in vitro. Acta Biomater 2012; 8: 3732-3743.
42. Seghir R, Arscott S. Extended PDMS stiffness range for flexible systems. Sens Actuators A Phys 2015; 230: 33-39.
43. Li S, Ding C, Guo Y, Zhang Y, Wang H, Sun X, et al. Mechanotransduction regulates reprogramming enhancement in adherent 3D keratocyte cultures. Front Bioeng Biotechnol 2021; 9: 1-15.
44. Wang Z, Wen F, Chong MSK. Surface Modification of Tissue Engineering Scaffolds. In: Gao C, editor. Polymeric Biomaterials for Tissue Regeneration: From Surface/Interface Design to 3D Constructs. Singapore: Springer Nature Singapore; 2023. p. 227-64.
45. Zhang Q, Lin S, Li Q, Zhao D, Cai X. Cellular response to surface topography and substrate stiffness. Cartilage Regen 2017: 41-57.
46. Wang ZY, Teoh SH, Johana NB, Khoon Chong MS, Teo EY, Hong MH, et al. Enhancing mesenchymal stem cell response using uniaxially stretched poly(ε-caprolactone) film micropatterns for vascular tissue engineering application. J Mater Chem B 2014; 2: 5898-5909.
47. Wang ZY, Teo EY, Chong MS, Zhang QY, Lim J, Zhang ZY, et al. Biomimetic three-dimensional anisotropic geometries by uniaxial stretch of poly(ε-caprolactone) films for mesenchymal stem cell proliferation, alignment, and myogenic differentiation. Tissue Eng Part C Methods 2013; 19: 538-549.
48. Zhu S, Hou J, Liu C, Liu P, Guo T, Lin Z, et al. An engineered tenogenic patch for the treatment of rotator cuff tear. Mater Des 2022; 224: 1-11.
49. Wang Z, Lee WJ, Koh BTH, Hong M, Wang W, Lim PN, et al. Functional regeneration of tendons using scaffolds with physical anisotropy engineered via microarchitectural manipulation. Sci Adv 2018; 4: 1-12.
50. Lü D, Luo C, Zhang C, Li Z, Long M. Differential regulation of morphology and stemness of mouse embryonic stem cells by substrate stiffness and topography. Biomaterials 2014; 35: 3945-3955.
51. Oh S, Brammer KS, Li YJ, Teng D, Engler AJ, Chien S, et al. Stem cell fate dictated solely by altered nanotube dimension. Proc Natl Acad Sci U S A 2009; 106: 2130-2135. 
52. Yoon JJ, Ismail S, Sherwin T. Limbal stem cells: Central concepts of corneal epithelial homeostasis. World J Stem Cells 2014; 6: 391-403.
53. de Paiva CS, Chen Z, Corrales RM, Pflugfelder SC, Li DQ. ABCG2 transporter identifies a population of clonogenic human limbal epithelial cells. Stem Cells 2005; 23: 63-73.
54. Li Y, Giovannini S, Wang T, Fang J, Li P, Shao C, et al. p63: A crucial player in epithelial stemness regulation. Oncogene 2023; 42: 3371-3384.
55. Liang SC, Yang CY, Tseng JY, Wang HL, Tung CY, Liu HW, et al. ABCG2 localizes to the nucleus and modulates CDH1 expression in lung cancer cells. Neoplasia 2015; 17: 265-278.
56. Chen LW, Chen YM, Lu CJ, Chen MY, Lin SY, Hu FR, et al. Effect of air-lifting on the stemness, junctional protein formation, and cytokeratin expression of in vitro cultivated limbal epithelial cell sheets. Taiwan J Ophthalmol 2017; 7: 205-212.
57. Gonzalez-Andrades M, Alonso-Pastor L, Mauris J, Cruzat A, Dohlman CH, Argüeso P. Establishment of a novel in vitro model of stratified epithelial wound healing with barrier function. Sci Rep 2016; 6: 1-9.
58. Tiwari A, Swamynathan S, Campbell G, Jhanji V, Swamynathan SK. BMP6 regulates corneal epithelial cell stratification by coordinating their proliferation and differentiation and is upregulated in pterygium. Invest Ophthalmol Vis Sci 2020; 61: 1-9.
59. Zhang L, Wang YC, Okada Y, Zhang S, Anderson M, Liu CY, et al. Aberrant expression of a stabilized β-catenin mutant in keratocytes inhibits mouse corneal epithelial stratification. Sci Rep 2019; 9: 1-9.
Iranian Journal of Basic Medical Sciences
Volume 28, Issue 7
July 2025
Pages 916-928

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