Table 4.

Genes and proteins implicated in LSC and niche modulation.

Gene	Reference	Function in LSC Niche
RUNX1, PAX6, SMAD3	[99]	Li et al. utilized chromatin accessibility assays and constructed transcription factor interaction networks to elucidate core transcription regulatory circuitries implicated in modulating LSC function. RUNX1 and SMAD3 were found to be important in maintaining the corneal epithelium, and shRNA knockdown of either RUNX1 or SMAD3 notably results in decreased Notch1 and PAX6 expression, which subsequently disrupts LSC phenotype and stemness. In summary, knockdown of either RUNX1 or SMAD3 causes LSCs to transition to keratinized epidermal-like cells. Through modulation of the epigenetic landscape, RUNX1, PAX6, and SMAD3 maintain corneal epithelium identity. RUNX1 is specifically implicated in histone acetylation that increases the transcription of LSC-specific proteins.
TP63, Jag1	[82,101]	Jagged 1 (Jag1) is a protein expressed in human limbal tissue that activates Notch signaling. Gonźalez et al. demonstrated that Notch signaling activation through a recombinant Jag1 ligand decreases the LSC population and drives LSCs towards a mature corneal epithelium phenotype. By arresting mitotic division in culture limbal epithelial cells, Jag1-mediated activation of Notch signaling decreases basal limbal epithelial cell division. Overall, these findings suggest that Jag1-mediated Notch activation decreases LSC stemness, downregulates p63, diminishes the LSC population, and promotes LSC differentiation to a more mature phenotype. Although Ma et al. demonstrated contrary results in 2007, Gonźalez et al. hypothesized this could be due to differing levels of Notch activation due to different delivery systems of Jag1 ligand.
ABCB5	[21]	ATP-binding cassette, sub-family B, member 5 (ABCB5) is a plasma-membrane protein found in humans. Ksander et al. demonstrated that transplanted ABCB5-positive LSCs can reconstitute the corneal epithelium in a mouse model of LSCD. Furthermore, ABCB5 knockout mice demonstrate enhanced LSC proliferation and apoptosis, ultimately resulting in loss of LSCs and perturbed corneal homeostasis characteristic of a LSCD-like phenotype. Overall, these findings suggest that ABCB5 modulates LSC quiescence and survival via anti-apoptotic signaling.
SOX9	[106]	Through transcription factor gene expression profiling, Menzel-Severing et al. suggested that SOX9 is one of the major transcription factors expressed by LSCs. SOX9 is observed in the cytoplasm of basal LSCs and in the nuclei of suprabasal and corneal epithelial cells, suggesting that shunting of SOX9 to the nucleus of LSCs is associated with increased differentiation and activation. Furthermore, increased expression and nuclear localization of SOX9 is found in LSCs undergoing clonal expansion. RNAi knockdown of SOX9 in vitro results in significant upregulation of stem cell and terminal differentiation markers with simultaneously downregulation of markers of progenitor cells. Thus, a delicate signaling balance is believed to exist between SOX9 and Wnt/ß-catenin signaling that determines the fate of LSC quiescence and differentiation. Cytoplasmic SOX9 expression seems to maintain quiescent LSCs, whereas controlled nuclear translocation may promote shunting of LSCs into TACs.
TSPAN7, SOX17	[103]	Li et al. utilized a scRNA-seq platform to identify subpopulations of LSCs that range from quiescent to actively proliferating and differentiating cells. Characterization of the changes in gene expression along this spectrum of quiescence identified TSPAN7 and SOX17 as novel markers of LSCs that may impact stemness and function. RNA silencing of both mRNA products inhibits cellular proliferation and perturbs corneal epithelial regeneration. Activation of these proteins is associated with increased progenitor cell marker expression. Thus, TSPAN7 and SOX17 may be markers of LSCs with the capacity to regenerate and repair corneal epithelium.
PBK, H2AX, ATF3	[22]	Kaplan et al. utilized scRNA-seq in wild-type and autophagy-deficient mice to characterize molecular differences between LSCs, mature TACs, and mature differentiated corneal epithelial cells. Autophagy-deficient mice exhibit altered expression of PBK, H2AX, and ATF3. Overall, autophagy was found to be a positive regulator of LSCs, promoting differentiation and reconstitution of corneal epithelium in wound healing. Autophagy promotes expression of PBK and H2AX, two proteins that seem to promote LSC differentiation and proliferation, and downregulates expression of ATF3, a transcription factor that seems to promote LSC quiescence. Further investigation of the role of ATF3 via siATF3 treatment demonstrated that downregulation of ATF3 results in increased cell growth compared with that of control siRNA-treated cells. An important implication of this work is the potential regulatory role of ATF3 in decreasing LSC proliferation and maintaining quiescence.
Wnt6	[97]	Bonnet et al. utilized 3T3 feeder cells with differential expression of Wnt6 to observe the dose-dependent effect of Wnt6 on LSC proliferation and differentiation. Co-culture of LSCs with supporting cells expressing high levels of Wnt6 results in increased proliferation of LSCs and decreased expression of differentiation markers. In addition to noncanonical Wnt/ß-catenin signaling, Wnt6 was also observed to activate noncanonical signaling in vitro. Bonnet et al. proposed that medium to high levels of Wnt6 expression are essential in promoting LSC self-renewal and stemness, thereby allowing for optimization and modulation of LSCs in culture.
Frizzled 7	[100]	Mei et al. utilized qRT-PCR and immunostaining to profile the expression of various Frizzled receptors in the human limbus, identifying Frizzled 7 (Fz7) receptor as predominantly expressed. Fz7 ligand colocalizes with other LSC markers and not with mature, differentiated corneal epithelium. shRNA knockdown of Fz7 results in significantly decreased expression of LSC markers, such as ABCG2, K14, and ΔNpP63α as well as significantly decreased colony forming efficiency. These results implicate the role of Fz7 in promoting LSC stemness and function.
Plk3	[108]	Wang et al. utilized a hypoxic stress culture platform to study the differential effects of the hypoxia-induced Plk3 signaling pathway on human LSCs and human corneal epithelial cells. Hypoxic conditions seem to promote LSC differentiation via downregulated Plk3 transcription, whereas hypoxic conditions have the opposite effect on mature corneal epithelial cells, resulting in upregulated Plk3 activity with subsequent apoptosis. This research suggests that downregulated Plk3 activity in LSCs as seen under hypoxic stress promotes LSC differentiation and prevents LSC apoptosis.