Pigmentation Is Associated with Stemness Hierarchy of Progenitor Cells Within Cultured Limbal Epithelial Cells

Abstract

Ex vivo cultured human limbal epithelial stem/progenitor cells (hLESCs) are the main source for regenerative therapy of limbal stem cell deficiency (LSCD), which is worldwide one of the major causes of corneal blindness. Despite many stemness-associated markers have been identified within the limbal niche, the phenotype of the earliest hLESCs has not been hitherto identified. We sought to confirm or refute the use of tumor protein p63 (p63) and ATP binding cassette subfamily B member 5 (ABCB5) as surrogate markers for hLESCs early within the limbal differentiation hierarchy. Based on a robust fluorescence-activated cell sorting and subsequent RNA isolation protocol, a comprehensive transcriptomic profile was obtained from four subpopulations of cultured hLESCs. The subpopulations were defined by co-expression of two putative stem/progenitor markers, the p63 and ABCB5, and the corneal differentiation marker cytokeratin 3. A comparative transcriptomic analysis yielded novel data that indicated association between pigmentation and differentiation, with the p63 positive populations being the most pigmented and immature of the progenitors. In contrast, ABCB5, either alone or in co-expression patterns, identified more committed progenitor cells with less pigmentation. In conclusion, p63 is superior to ABCB5 as a marker for stemness. Stem Cells 2018;36:1411-1420.

Isolation and enrichment of melanocytes from human corneal limbus using CD117 (c-Kit) as selection marker

“…Hayashi and coworkers reported that N-cadherin-positive LMs co-localized with CK15/N-cadherin-positive limbal basal epithelial cells and that N-cadherin-mediated cell–cell interactions are important in maintaining the potency of the epithelial stem cells 7 . LMs are enriched in limbal crypts which also harbor the most pigmented and immature LEPCs 5 , 8 . LMs from highly pigmented limbal biopsies were shown to support the stemness state and growth of LEPC and to promote the formation of multi-layered epithelial cell sheets 5 .…”

Understanding cornea epithelial stem cells and stem cell deficiency: Lessons learned using vertebrate model systems

“…The harvested tissue can be tested for the presence of CESCs using various markers (Rama et al, 2010). The transcription factor p63 is a commonly used marker, but detects all proliferative cells (CESCs and TACs) (Arpitha, Prajna, Srinivasan, & Muthukkaruppan, 2008; Di Iorio et al, 2005; L. Liu et al, 2018; Pellegrini et al, 2001). Studies are still ongoing to find suitable markers to identify and isolate pure populations of CESCs for transplantation (Pajoohesh‐Ganji, Pal‐Ghosh, Tadvalkar, & Stepp, 2012; Sonam et al, 2019), and a specific marker to differentiate stem cells from TACs would be very beneficial (Sacchetti et al, 2018).…”

“…The harvested tissue can be tested for the presence of CESCs using various markers (Rama et al, 2010). The transcription factor p63 is a commonly used marker, but detects all proliferative cells (CESCs and TACs) (Arpitha, Prajna, Srinivasan, & Muthukkaruppan, 2008;Di Iorio et al, 2005;L. Liu et al, 2018;Pellegrini et al, 2001).…”

Generation of TGFBI knockout ABCG2+/ABCB5+ double-positive limbal epithelial stem cells by CRISPR/Cas9-mediated genome editing

“…ABCB5 was mainly expressed in basal layer cells of the mouse limbus. In human eyes, ABCB5 + cells were located in the basal layer of the limbus and co-expressed ΔNp63α− a known expressed in epithelial stem cells [16, 17], including human limbal stem cells[18, 19].…”