Current perspectives of limbal‐derived stem cells and its application in ocular surface regeneration and limbal stem cell transplantation

Abstract Limbal stem cells are involved in replenishing and maintaining the epithelium of the cornea. Damage to the limbus due to chemical/physical injury, infections, or genetic disorders leads to limbal stem cell deficiency (LSCD) with partial or total vision loss. Presently, LSCD is treated by transplanting limbal stem cells from the healthy eye of the recipient, living‐related, or cadaveric donors. This review discusses limbal‐derived stem cells, the importance of extracellular matrix in stem cell niche maintenance, the historical perspective of treating LSCD, including related advantages and limitations, and our experience of limbal stem cell transplantation over the decades.


| INTRODUCTION
Stem cells have the self-renewal ability and potency to differentiate into specific cell types or an entire organism. Adult stem cells (ASCs) are populations of cells involved in an internal repair mechanism generating replacement for cells lost through the wound healing process, wear and tear, injury, and diseases. 1 They reside at specific anatomical locations and may remain quiescent for long periods until activated by their need to maintain tissue homeostasis. 2 Stem cells for the cornea are present at the corneoscleral limbus. Two population of ASCs exists in the limbal niche, epithelial and stromal stem cells, referred as limbal epithelial stem cells (LESCs) 3 and corneal stromal stem cells (CSSCs), 4 respectively. These stem cell populations are essential to maintain corneal transparency. 5 Additionally, the limbal niche also harbors early transient amplifying cells, melanocytes, and Langerhans cell. [6][7][8] As per the recent global consensus by the International Limbal Stem Cell Deficiency Working Group, they concluded that "Autologous limbal stem cell transplantations using the least amount of donor tissue, such as simple limbal epithelial transplantation (SLET), ex vivo-cultivated autologous LSC, and modified conjunctival limbal autograft (CLAU), are preferred over other surgical treatments for unilateral or subtotal bilateral limbal stem cell deficiency (LSCD) or whenever feasible because of better long-term outcomes and fewer complications". 9 This review summarizes the recent significant progress based on our experience and literature survey on ocular surface stem cells, their culture and expansion, corneal epithelial regeneration, limbal stromal cells, and the role of limbal niche. We have also discussed the associated challenges and need of various stem cells in ocular therapies.

| CULTURE AND EXPANSION OF LIMBAL-DERIVED STEM CELLS
Limbal stem cells are the well-characterized cells of the ocular surface 10 other than conjunctival progenitors whose anatomical location remains elusive. 11 LESCs and CSSCs can be admirably identified in vitro with different morphological and slow-cycling properties of the cells. 12 Limbal epithelial cultures used for transplantation in treating ocular surface burns are known to contain stem cells that are identified by the positive expression of the p63α 13 and ABCG2. 14 The recent discovery of ABCB5 as a marker for limbal stem cells and its tendency to localize with p63α could aid in exclusive isolation of stem cell populations in limbus. 15 The properties of LESC were further retained or enhanced by their culture on feeder layers that includes gamma-irradiated 3T3 cells, human embryonic fibroblasts, or human amniotic epithelial cells. Interestingly, intrinsic feeder layers were also observed in limbal epithelial cultures that help in the maintenance of the stemness in the limbal explant culture system. 16,17

| Limbal epithelial cells
In vitro cell expansion potential of the limbal tissues had revealed that tissue obtained from living subjects had a higher capability to initiate cell growth than the preserved cadaver tissues. 18 This decrease in cell viability of the cadaver tissues could be attributed to preservation outside their nativity. Whereas better cell growth with fresh tissue than cadaver tissue is comprehensible, it seems that limbal stem cells remain viable in the limbal niche during storage of cadaver corneal tissue for weeks. 19 We had also observed from our study that 60% of the cadaver tissues were able to initiate the cell growth in contrast to 90% of the live tissues. Interestingly, these cadaver tissues that have initiated cell growth compete with the live tissue in terms of cell expansion and epithelial cell integrity, demonstrating viable limbal stem cell populations despite preservation. Moreover, the epithelial cell sheets developed from the limbal explants ex vivo showed greater integrity, as evident from the rich development of desmosomes, hemidesmosomes, and E-cadherins. 20,21 Furthermore, a study reported by Ekpo et al had cultured the cadaver tissues adjacent to the limbus, that is, toward corneal (L.cor) and conjunctival (L.conj) sides. Interestingly, they have noted that the cells cultured from the L.conj had more growth potential and stemness than L.cor, as evident from p63α expression. 22 Though live limbal tissue has desirable cell growth, cadaver limbal rims obtained either during corneal keratoplasties or from stored eye banking tissues also serves as a viable alternative for transplantation purposes.

| Limbal niche cells
CSSC exhibits mesenchymal stem cell-like properties, 23 although they are of neurocrest origin and are identified as side population cells expressing ABCG2 and PAX6. While CSSCs are multipotent mesenchymal cells, LESCs are confined to deriving corneal/limbal epithelium. 4 Though the primary culture of CSSC survives longer passages (40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50), they may lose their plasticity with the increase in senescence and therefore are preferred at earlier passages in cell therapies. 24 Our in vitro experiments of limbal cultures had shown that these cells are predominantly identified as a streak at the expanding edge of the expressing ABCG2 ( Figure 1A 25 Recent studies have shown that fiber hydrogel and serumfree media synergize to provide an optimal environment for keratocyte phenotype growth as well as the regeneration of damaged corneal stroma. 26,27 Interestingly, stromal cells cultured on twodimensional (2D) silk fibroin sheets and stacked to provide threedimensional (3D) structure leverages both 3D microenvironment and stromal cell sufficiency. 28

| EXTRACELLULAR MATRIX NICHE FOR ENHANCED CELL THERAPY
The extracellular matrix (ECM) harboring ASCs comprises adhesive proteins, proteoglycans, polysaccharides, and structural proteins: fibronectin, laminin, and tenascin-c. The shape and composition of ECM depend upon the tissue, developmental stages, and pathological conditions, thereby affecting the overall fate of the stem cells. ECM orchestras the native 3D environment surrounding ASCs, and neighboring cells and also serves as an active reservoir of soluble factors, thereby modulating cellular behavior. 29 Besides being a dynamic ing artificial corneas or scaffolds to support stem cell maintenance. 31 Additionally, replacement of damaged ECM with AM or bioengineered ECM would provide enhanced cell therapies. 32,33 Therefore, it is essential to understand the ASC-ECM interaction in maintaining niche, which provides a conducive milieu for in vitro expansion of ASCs. 34,35 Understanding the specific components of ECM, its role in regulating stem cell behavior in different tissues can be deciphered from the in vivo studies and with the use of engineered in vitro niche. 36 Similarly, ECM is vital for the physiological renewal of the ocular surface/limbal microenvironment and plays a crucial role in the function and maintenance of the limbal niche. 37 The limbal niche comprises well-organized ECM, signaling drivers, and niche cells such as melanocytes, immune cells, vascular cells, nerve cells, and stromal cells, which are compromised in certain hereditary conditions or severe insults to the limbus. 32,[38][39][40][41] Bioengineered corneas are fabricated using polyethylene glycol to generate a niche like structure 42 or entire corneal stroma with keratocytes embedded in them. 43 Porous hydrogels similar to collagen structure were also developed by the Griffith's group that amalgamates with natural fibrin compound to form LiQD corneas. 44 These studies will potentially open new avenues for creating more accurate in vivo niche features by applying in vitro system, enabling a better understanding of the biology and efficacy of stem cell-based therapies.

| HISTORICAL PERSPECTIVE AND PROGRESSIVE SHIFT IN LIMBAL STEM CELL TRANSPLANTATION
The concept of limbal stem cells in treating LSCD was clinically applied by Kenyon and Tseng, where the conjunctiva with limbus tissue was used for ocular surface reconstruction in patients with chemical injuries. 45 Since then, chemical burns of the eye had been challenging with complexity and variation among the patients. Much emphasis has been given to understand the extent of LSCD firsthand before deciding on the therapeutic approach. Clinical presentation of ocular surface chemical burns has been graded using either Roper-Hall's or Dua's classification to understand the extent of limbal tissue damage. 46 With further clinical advancements, in vivo confocal  With the advent of CLET and SLET, several other cell sources are also being explored in treating LSCD. Specifically, induced pluripotent stem cells (iPSCs) offer a promising approach to generate mature corneal/limbal epithelium with 3D corneal organoids. 74 Once standardized, iPSC-derived corneal/limbal epithelial cells could be an unlimited source for limbal cell therapies. However, all these techniques have their advantages and limitations (Table 1) and are used depending on the condition of the patient. Moreover, these cells were also capable of reversing the already existing scars into a clear cornea, as evident in nitrogen injured scar models of mice. 83 Interestingly, the collagen reorganization of the wounded mice cornea and the lamellar structure was indistinguishable from the normal cornea making the limbal stromal cells a potent therapeutic for treating corneal stromal blindness. 84 The immunomodulation ability of limbal stromal-derived cells makes them an excellent source for treating corneal scars and acute chemical injuries of the cornea.

CONFLICT OF INTEREST
The authors declared no potential conflicts of interest.

AUTHOR CONTRIBUTIONS
V.S.: conception and design, financial support, administrative support, collection and/or assembly of data, provision of study material or patients, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript. A.T., A.R.K.: collection and/or assembly of data, data analysis and interpretation, manuscript writing. V.S.S.: conception and design, financial support, administrative support, provision of study material or patients, data analysis and interpretation, manuscript writing, final approval of manuscript.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.