Javid D, Zhang J, Alamdari DH. Hot Topics in Limbal Stem Cells Culture: Mini Review. J Ophthalmol Sci. 2021;3(1):1-5

Mini Review Article Open Access

Hot Topics in Limbal Stem Cells Culture: Mini Review Dina Javid1, Jie Zhang2, Daryoush Hamidi Alamdari3* 1Department of Clinical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 2Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand 3Surgical Oncology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.

Article Info Abstract

Article Notes Received: November 16, 2020 One of the most effective treatments for Limbal Stem Cells Deficiency Accepted: January 06, 2021 (LSCD) is Cultivated Limbal Epithelial Stem Cells (CLET). Precise knowledge of limbal stem cells culture increase the quality of treatment, mitigate the *Correspondence: side effects and complications of organ transplantation, and expedite the *Daryoush Hamidi Alamdari, Surgical Oncology Research Center, treatment process. However, protocols for CLET need further optimization and Mashhad University of Medical Sciences, Mashhad, Iran; Email: [email protected]. standardization. This mini review covers basic concepts and important criteria that should be taken into consideration in designing a successful CLET that can ©2020 Alamdari DH. This article is distributed under the terms of be helpful for other targets of regenerative medicine. the Creative Commons Attribution 4.0 International License.

Keywords: Limbal stem cells Limbal Epithelial Stem Cells (LESCs) refer to unipotent adult CLET stem cells (ASCS) capable of extensive self-regeneration. LESCs play Substrate

anti-angiogenic and anti-inflammatory roles in fostering the integrity of the whole corneal surface and inhabiting at limbus. Limbus is made of a profound and complicated network of crypts enclosed by the palisades of Vogt, which creates a niche for corneal epithelial stem cells. During the normal corneal epithelial performance, LESCs stay in the limbus where they are split asymmetrically, forming the first generation of novel basal corneal epithelial cells – transient amplifying cells (TACs) in the peripheral . They are more activated over episodes of1 considerable wound healing where the corneal surface regeneration comprises division, migration, and maturation of these cells . However, it seems that the shape and dimensions of a corneal epithelial injury determine the involvement of LSCs in the wound healing process as2 it has been shown in a mouse study that smaller circular wounds with 0.75-mm diameter do not rely on LSCs as do larger wounds . Given their engagement orin thegenetic regeneration factors. of healthy epithelium, LESCs are utilized for the treatment of degeneration induced by external Limbal stem cells markers

could be characterized using markers or mechanical properties. Negative markers includes molecules expressed in differentiated epithelium (cytokeratins (CK3, CK12), conjunctival epithelium3 (CK5, CK14, CK19), transitional cells (Vimentin), transient amplifying cells (α9 and β1 integrins, α‐enolase, and connexin‐43) . Positive markers include ΔNp63α, C/EBPσ, Bmi1, ATP-binding cassette transporter (ABCG2), ATP- binding cassette, sub-family B, member 5 (ABCB5) and Notch‐1. The nuclear transcription factor of ΔNp63α, is a major factor affecting the proliferative capacity of stem cells in stratified epithelia. In cultures where more than 3% are accounted4 for by p63, the rate of transplantation rate success is near 80% . Both C/EBPσ and Bmi1 Page 1 of 5 Javid D, Zhang J, Alamdari DH. Hot Topics in Limbal Stem Cells Culture: Mini Review. J Ophthalmol Sci. 2021;3(1):1-5 Journal of Ophthalmological Science

. are believed to regulate LSC senescence, and the expression5 been successful in the treatment of partial, unilateral, of them is downregulated in response to corneal injury or total LSCD. This technique is characterized by rapid Research suggests that ABCG2+ cells6 are not linked to epithelialization, little inflammation, and the requirement elevated colony-creating efficiency . Notch‐1 has7 been of little donor tissue. The general success14 rate of this recognizedand regeneration in a subclass8 of ABCG2‐expressing cells . ABCB5 process is considered to be 70%–80% . Concisely, limbal is required for corneal development, LESC homeostasis epithelial cells are harvested through a small limbal biopsy. . Recently, mechanical properties as LESCs, located in the limbus, are isolated and cultured phenotypic and label-free markers such as cell stiffness in a laboratory to produce a sheet of cultured LECs that and size have been in the spotlight as stemness indicator in is suitable for transplantation onto the cornea. EMA the . It has been proved that LSCs have small approved the use of ex vivo-expanded autologous human diameter and high nucleus/cytoplasm ratio in comparison orcorneal severe epithelial burn-related cells containingLSCD. transplantation with other cells from the limbus. In addition, they are larger (Holoclar®) in 2015 as a cure for patients with moderate than central cornea cells but smaller than differentiated Sources of tissue for cultivating limbal stem cells may be LECslimbal. Because epithelial using cells antibodies (LECs) andto identify considerably LSCs in softer vitro than 9 both the central cornea cells and the differentiated patients (autograft). Accordingly, it often comes from the healthy eye of the patient’s in unilateral cases, is inconvenient, time consuming and cellular physiology relative in bilateral cases or from animals (xenograft). from another individual (allograft), from a cadaver or living can be subsequently affected, label-free cell-enrichment approaches alongside antibody-based methods can greatly Studies demonstrate that changes in the elasticity of develop LSCD treatment by enabling a faster and cheaper human cornea have an effect on the cellular behavior of processLimbal to identify Stem Cellstem-like Deficiency cells. (LSCD) is a condition outcome LSCs. Hence,15 younger donor age is associated with better . According to recent research, the success rate arising from the inadequate quantity and/or dysfunction of allogeneic and autologous16 CLET is not significantly of LSCs. It is characterized by conjunctivalization and different in patients with LSCD . The most recent advances inflammation of corneal surface and neovascularization in therapeutic methods suggest that epithelial cells could differentof corneal categories stroma. This for mayLSCD result etiology: in pain, primary low vision, and be harvested from non-ocular sites including oral mucosal photophobia, and finally corneal blindness. There are epithelial cells, conjunctival epithelial cells, hair follicle bulge-derived epithelial stem cells, amniotic epithelial secondary causes, acquired immune-related, non- cells, human embryonic stem cells, induced pluripotent immune and hereditary causes, and more unfavorable and stem cells, umbilical cord lining epithelial stem. cells and favorable. Unilateral LSCD is primarily caused by chemical Wharton’s Jelly mesenchymal stem cells,17 mesenchymal Methods of Limbal Stem Cells Expansion injury, which accounts for 66–75%. Unilateral of cases. LSCD One is majormore stem cells, immature dental pulp stem cells cause of bilateral LSCD is Stevens–Johnson10 syndrome/toxic Explant culture or cell suspension culture epidermal necrolysis (SJS/TEN) 11. prevalent than bilateral LSCD. It commonly affects young . In the cell maleTreatment subjects, with of Limbal developing Stem total Cell LSCD Deficiency (LSCD) suspension culture, the biopsy specimen derived from the limbal area is disassociated into separate cells by enzymes including trypsin, dispase, and collagenase, then seeded on is a function of the extent of the limbus’ involvement a substrate carrier to be further transferred to the ocular (sectoral vs total) as well as the unilateral or bilateral surface. In the explant culture, it is customary to put the nature of the disease. With regard to partial LSCD, there explant tissue on a substrate, which enables the growth are a number of conservative nonsurgical alternatives, and proliferation of cells on the substrate surface. Findings including autologous serum drops, therapeutic soft contact suggest that explants from the Lconj (located outermost18 lens, therapeutic scleral lens, topical lubrication as well as12 scraping and amniotic membrane transplantation (AMT) . adjacent to the conjunctiva) and Lm (middle2 limbus) sites some conservative surgical alternatives such as corneal for live explant should be selected2 for cadaveric for limbal explant cell expansion for CLET . The graft size should be a minimum of 0.319 mm Regarding the total ocular surface diseases, the latest and 0.5 mm . Furthermore, organ treatment includes the application of cultivated corneal culture systems require a bio-adhesive to improve the limbal epithelial cell transplantation. It encompasses adhesion of the cultivated cells on the carrier. Apparently, conjunctival limbal autograft(CLAU), conjunctival limbal and yy etc the best bio-adhesive4 is fibrin glue (FG), compared to xx allograft (CLAL), Keratolimbal allograft (KLAL),13 simple . . Cell suspensions formed from fresh tissue is limbal epithelial transplantation (SLET) and ex vivo more likely to provide a larger and more viable stem cell cultivatedCultivated limbal Limbal epithelial Epithelial stem cells Stem (CLET) Cells (CLET) population than that obtained20 from organ culture–stored has human limbal epithelium . However, organ-culture Page 2 of 5 Javid D, Zhang J, Alamdari DH. Hot Topics in Limbal Stem Cells Culture: Mini Review. J Ophthalmol Sci. 2021;3(1):1-5 Journal of Ophthalmological Science conditions can preserve limbal cell mitotic potential if ve expanded31 limbal tissue is excised early after circulatory arrest21. ha . Among all the xenobiotic-free conditions 2D or 3D culture techniques. Use of LSCs for clinical tested, modified supplemented hormonal32. epithelium medium (mSHEM) was the most efficient and consistent in Diverse substrates and carriers applications requires a high quality and quantity of cells. supporting LSC phenotype and growth This requires large-scale expansion of LSCs followed by have been presented efficient and homogeneous differentiation into functional to cultivate limbal stem cells in an attempt to improve the results. Traditional approaches for preservation and clinical outcomes. The long-run viability of donor LSCs expansion of cells rely on two-dimensional (2-D) culturing could be undermined by the absence of a supporting and methods using plastic culture plates and xenogeneic media. and cells tend to lose clonal and differentiation capability healthy LSC niche. The solutionin vivo is adopting an optimal These methods provide limited expansion as a monolayer substrate to mimic the mechanical properties and transparency similar to that of which may be solid, upon long-term passaging. To address the various problems softsemi-solid substrates or liquid. in order LSCs to maintainniche is twice stemness as soft33 as adjacent facing 2-D culture of SCs, three-dimensional (3-D) culture tissue of the cornea and sclera , as stem cells require very methods have been developed. Natural 3-D niches allow for . In addition, interactions between cells, inclines of nutrients, oxygen, in it has been shown that smooth surfaces with pore sizes and waste. Recently, new approaches for the expansion vivo <1 µm stimulated corneal epithelial cell migration, while34 of LSCs have emphasized 3-D cell growth to mimic the rough surfaces with pore sizes >1 µm lessened migration . (3D-CC-BM) environment,22 including, 3D culture system of clusters and discontinuous shear flow induced differentiation, but35 of LSCs on bone marrow-derived23 mesenchymal stem cells constant shear flow preserved the stemness of LSCs (3D LaBP) , Real Architecture For 3D Tissue and tissue Consequently, as studies demonstrate, the phenotype of equivalents24 (RAFT-TEs) , laser-assisted 3D bioprinting LESCs is highly36 dependent on the mechanical properties of , cultivation of limbal tissue explants for25 their substrate , biochemical aspects of chosen substrate more than 3 month in medium without and 3D using culture scaffolds system, should be taken into consideration including, elastic 3Dof limbal culture tissue system explant of limbal on amniotic epithelial26 membrane cells on limbalusing mesenchymal cells (3D CC-LMC) properties, surface topography, porosity and shear flow as 27 nanotopographic feature. platelet-rich plasma-Fibrin glue (3D AM-PRP-FG) . The 2D Furthermore, it is essential that the carrier system culture lasts for about 2 weeks, which could be extended can be degraded in the body. Such carriers include: (i) using the air-lifting28 technique. In this method, the culture is cell-free biological materials22 including, modified human subjected to the air-liquid interface to produce a multilayer amnioticcells (PLA) membranes, Fibrin, Natural Collagen, Chitosan- epithelium . However, through constructing. 3D scaffold to gelatin, Keratin,37 PRP 38, Human Processed Lipoaspirate decrease exposure to the oxygen as an 27air-lifting technique, Epithelial basement membrane of human39 the Xenogeneicculture length can or remain xenogeneic-free. 2 weeks decellularized cornea and Descemet’s membrane Traditionally, (ii) plastics that are compatible with the body such LSCs often require undefined or xenogeneic materials as, plastic compression collagen, siloxane hydrogel including attachment substrates such as feeder layer contact lenses, poly(-caprolactone), polymethacrylate, of mouse 3T3 fibroblasts, culture medium containing hydroxyethylmethacrylate, and (iii) physiologically xenogeneic cytokines and growth factors, as well as degradable synthetic polymers and biopolymers such fetal bovine serum (FBS). Use of xenogeneic or animal severe immunologic reactions in recipients and limit as chemically crosslinked collagen,. silk fibroin, human derived media can potentially transmit pathogens, cause anterior lens capsule, poly (ethylene40 glycol), poly (vinyl alcohol), poly(lactide-co-glycolide) reproducibility between cultures due to lot-to-lot variation of the material used. Furthermore, studies have shown Human amniotic membrane is presently the most that the composition and organization of the extracellular prevalent substrate in clinical applications of CLET, but it matrix have important roles in cell differentiation29 through suffers from a number of drawbacks such as a theoretical sending biochemical and mechanical signals . The U.S. buildperil more of infection, accurate and biological functional instability, replicates of and native lack tissue of useFood of and xenogeneic Drug Administration cells during (FDA) production have imposed of tissue strict. standardized preparation protocols. Therefore, lately, to guidelines against xenobiotic-transplantations and the30 structure, more synthetic scaffolds have been developed as Subsequently, due to recent concertation on xenobiotic-an- scaffoldsthey resemble physical and chemical properties40 of tissue and AMfree and culture autologous systems, PRP utilization and xenobiotic-free of autologous culture serum media like ECMnovel including,collagen41 scaffolds microfabricated. scaffolds , electrospun42 human serum, substrate such as autologous FG, hum , synthesized43 carboxymethyl cellulose

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