Abstract:Human Corneal epithelial stem cells (CESCs) have been identified to reside in limbus for more than 2 decades. However, the precise location of CESCs in other mammalian remains elusive. This study was to identify differential localization of putative CESCs in mice. Through a series of murine corneal cross-sections from different directions, we identified that anatomically and morphologically the murine limbus is composed of the thinnest epithelium and the thinnest stroma without any palisades of Vogt-like niche… Show more
“…In bromodeoxyuridine (BrdU) pulse-chase experiments, most label-retaining cells (LRCs) were confined to the limbus, irrespective of age ( Figure S2 ). However some LRCs occupied the central cornea, similar to a recent report ( Li et al., 2017 ). Figure 3 K14 + Progenitor Cell Distribution in the Corneal Epithelium of Confetti Mice during Aging (A, C, E, and G) Whole flat-mount corneas from Confetti mice from 2-, 10-, 20-, and 60-week-old mice 1 week post TAM (n = 3/time point); only the RFP channel is displayed.…”
Section: Resultssupporting
confidence: 91%
“…Furthermore, BrdU pulse-chase experiments demonstrated that LRCs were predominantly confined to the limbus regardless of age ( Figure S2 ). Notably, some LRCs were detected in the central cornea and persisted even in older mice, a result corroborated by others ( Li et al., 2017 ). However, a longer chase period may have diluted the label to a point where these cells are no longer detectable.…”
SummaryThe dynamics of epithelial stem cells (SCs) that contribute to the formation and maintenance of the cornea are poorly understood. Here, we used K14CreERT2-Confetti (Confetti) mice, sophisticated imaging, and computational modeling to trace the origins and fate of these cells during embryogenesis and adult life. We show that keratin-14 (K14+)-expressing progenitors are defined and widely distributed across the E16.5 cornea, after which they undergo cycles of proliferation and dispersal prior to eyelid opening. K14+ clonal patches disappear from the central cornea and are replaced by limbal-derived K14+ streaks, a finding that aligned with bromodeoxyuridine label-retaining studies. We also elucidated the mechanism by which SC clones are lost during life and propose this is due to population asymmetry and neutral drift. Finally, we established that the occurrence of an equatorial migratory mid-line is a consequence of apoptosis in a narrow nasal-temporal region, the site where eyelids meet during blinking.
“…In bromodeoxyuridine (BrdU) pulse-chase experiments, most label-retaining cells (LRCs) were confined to the limbus, irrespective of age ( Figure S2 ). However some LRCs occupied the central cornea, similar to a recent report ( Li et al., 2017 ). Figure 3 K14 + Progenitor Cell Distribution in the Corneal Epithelium of Confetti Mice during Aging (A, C, E, and G) Whole flat-mount corneas from Confetti mice from 2-, 10-, 20-, and 60-week-old mice 1 week post TAM (n = 3/time point); only the RFP channel is displayed.…”
Section: Resultssupporting
confidence: 91%
“…Furthermore, BrdU pulse-chase experiments demonstrated that LRCs were predominantly confined to the limbus regardless of age ( Figure S2 ). Notably, some LRCs were detected in the central cornea and persisted even in older mice, a result corroborated by others ( Li et al., 2017 ). However, a longer chase period may have diluted the label to a point where these cells are no longer detectable.…”
SummaryThe dynamics of epithelial stem cells (SCs) that contribute to the formation and maintenance of the cornea are poorly understood. Here, we used K14CreERT2-Confetti (Confetti) mice, sophisticated imaging, and computational modeling to trace the origins and fate of these cells during embryogenesis and adult life. We show that keratin-14 (K14+)-expressing progenitors are defined and widely distributed across the E16.5 cornea, after which they undergo cycles of proliferation and dispersal prior to eyelid opening. K14+ clonal patches disappear from the central cornea and are replaced by limbal-derived K14+ streaks, a finding that aligned with bromodeoxyuridine label-retaining studies. We also elucidated the mechanism by which SC clones are lost during life and propose this is due to population asymmetry and neutral drift. Finally, we established that the occurrence of an equatorial migratory mid-line is a consequence of apoptosis in a narrow nasal-temporal region, the site where eyelids meet during blinking.
“…Earlier attempts to clarify the developmental biology and differentiation hierarchy of hLESCs have been hampered by the use of nondiscovery based methods such as microarray analysis , poor study material such as nonhuman or whole unfractionated tissue , or impure hLESC cultures . A few discovery based next‐generation sequencing hLESCs transcriptomic studies have been conducted but these were also limited by the use of in situ material or nonhuman models .…”
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.
“…In addition, the corneal epithelial cells exhibited the ability to undergo serial transplantation, suggesting that self-renewing SCs exist in the entire corneal epithelium. The analysis of K14 CreER -based lineage tracing, transplantation and culture experiments also support the existence of SC/progenitor population in the central or entire cornea (Li, et al, 2017;Amitai-Lange, et al, 2015). However, lack of lineage tracing tools to specifically mark epithelial subpopulations and definitive regional markers hampered us to determine the SC identity of the corneal epithelium.…”
Adult tissues contain label-retaining cell (LRC)s, which are relatively slow-cycling and considered to represent a unique property of tissue stem cell (SC)s. In the ocular surface epithelium, LRCs are detected in the limbus, a boundary between cornea and conjunctiva, and the fornix of the conjunctiva; however, the character of LRCs and identity of SCs remain unclear due to lack of appropriate molecular markers. Here we show that the ocular surface epithelium accommodates spatially distinct stem/progenitor populations with different cell division frequency. By combining EdU pulse-chase analysis and lineage tracing with three CreER transgenic mouse lines: Slc1a3 CreER , Dlx1 CreER and K14 CreER , we detect distinct dynamics of epithelial SCs in the cornea and conjunctiva. In the limbus, long-lived SCs are labeled with Slc1a3 CreER and they either migrate centripetally toward the central cornea or laterally expand their clones within the limbal region. In the central cornea, cells are mostly non-LRCs, labeled by Dlx1 CreER and K14 CreER , and the number of clones declines after a short period of time with rare long-lasting clones, suggesting their properties as short-lived progenitor cells. In the conjunctival epithelium, which consists of bulbar, fornix and palpebral conjunctiva, each territory is regenerated by compartmentalized, distinct SC populations without migrating one region to another. The severe damage of the cornea leads to the cancellation of SC compartments, causing conjunctivalization of the eye, whereas milder limbal injury induces a rapid increase of laterally-expanding clones in the limbus. Taken together, our work provides lineage tracing tools of the eye and defines compartmentalized, multiple SC/progenitor populations in homeostasis and their behavioral changes in response to injury.
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