Immunofluorescence Tomography of Mouse Ocular Surface Epithelial Stem Cells and Their Niche Microenvironment

Abstract

PURPOSE. Currently, there are no definitive immunomarkers for epithelial stem cells (corneal and conjunctival) or their poorly understood niche microenvironment. The H2B-GFP/K5tTA mouse enables visualization of label-retaining cells (LRCs), which exhibit the functional marker of stem cell quiescence. We used immunofluorescence tomography to evaluate putative stem cell markers and LRCs of the mouse ocular surface.METHODS. H2B-GFP/K5tTA mice were pulsed for 56 days and then chased with doxycycline to label LRCs. Limbus and eyelid tissue was 3-dimensionally (3-D) reconstructed using immunofluorescence tomography to identify and characterize LRCs using the putative stem cell markers sox9, keratin 19, lrig1, blimp1, and abcb5.RESULTS. After 28 days of chase, LRCs were localized to the entire limbus epithelium and, infrequently, the anterior limbal stroma. Label-retaining cells comprised 3% of limbal epithelial cells after 56 days of chase. Conjunctival LRCs were localized to the fornix and comprised 4% of the total fornix epithelial cells. No stem cell immunomarker was specific for ocular surface LRCs; however, blimp1 enriched for limbal basal epithelial cells and 100% of green fluorescent protein-positive (GFP þ ) cells at the limbus and fornix were found to be lrig1-positive.CONCLUSIONS. Label-retaining cells represent a larger population of the mouse limbus than previously thought. They decrease in number with increased doxycycline chase, suggesting that LRC populations with different cell cycle lengths exist at the limbus. We conclude that current immunomarkers are unable to colocalize with the functional marker of epithelial stem cell quiescence; however, blimp1 may enrich for limbal epithelial basal cells.

Role of Human Corneal Stroma-Derived Mesenchymal-Like Stem Cells in Corneal Immunity and Wound Healing

“…The amount of ABCG2 + cells in in vitro LESC cultures has been indeed controversial; there is a big variance between the used methods 25 26 27 ; 2–4% of the LESCs cultured on mice feeder cells were found positive 28 , while the same amount could be reached with keratocyte medium cultivation as well 25 (similar to our cultured CSMSCs), and in other model, the cell suspension and explant showed 50–85% positivity 27 . Interestingly, another ATP-binding cassette transporter (ABCB5) has been suggested to be an in situ specific LESC marker 29 30 , however, in our dataset, neither the in vitro cultured LESCs nor the CSMSCs expressed ABCB5. Interestingly, a weak expression could be detected only in one of the BMMSC donors (see Supplementary Fig.…”

Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells

“…Sox9 has also been previously identified in limbal epithelial cells by microarray analysis of scraped epithelial samples 44 and transcriptome analysis of microdissected limbal epithelial crypts 37 as well as in cultivated human limbal epithelial keratinocytes 45 , but was not further analysed in situ . In addition, Sox9 has been identified as a marker of slow-cycling corneal epithelial stem cells in mouse eyes 46 , 47 . In extending this anecdotal evidence for a role of Sox9 in the human limbal stem cell niche, the present study demonstrated a striking differential sub-cellular localization of Sox9 in basal LEPC clusters and their progeny: Whereas LEPCs showed mainly cytoplasmic staining for Sox9, indicative of protein synthesis, suprabasal limbal and corneal epithelial cells showed exclusively nuclear localization suggestive of TF activity.…”

FACS-sorted putative oogonial stem cells from the ovary are neither DDX4-positive nor germ cells

“…Prdm1 is the key regulator of primordial germ cells (PGCs) in the embryo39 but it is also linked to stem cell pluripotency and maturation in non-germline cells404142434445464748. Here it was not a specific marker of the germline as it could be detected in dermal fibroblasts.…”

“…Only Prdm1 and Iftm3 , often regarded as germline markers 38 , were present in freshly isolated ovarian cells following FACS with DDX4 C25 antibody. Prdm1 is the key regulator of primordial germ cells (PGCs) in the embryo 39 but it is also linked to stem cell pluripotency and maturation in non-germline cells 40 41 42 43 44 45 46 47 48 . Here it was not a specific marker of the germline as it could be detected in dermal fibroblasts.…”