Abstract
PurposeWe investigate human lacrimal gland tissue to determine the presence of progenitor cells in this adult human tissue.MethodsSix human lacrimal gland tissues from donors were collected and stored immediately in the culture medium at 4°C until the next procedure. One part of the lacrimal gland tissue was prepared for immunofluorescence staining and the other part was prepared for primary cell culture. Immunofluorescence analysis was conducted to evaluate cultured lacrimal epithelial phenotype and progenitor cell markers for five passages. Real-time polymerase chain reaction (PCR) was performed to assess proliferation markers in the different passages. Three-dimensional culture and PCR were conducted to determine the differentiation potential of cultured human lacrimal gland cells.ResultsHuman lacrimal gland tissue expressed a number of epithelial progenitor cell markers. Precursor cell markers C-Kit, K15, Nestin, and P63 were observed in lacrimal gland tissues. Lacrimal gland epithelial cells were cultured successfully and passaged to P5. The cultured lacrimal gland epithelial cells were positive for pan-cytokeratin (PCK), AQP5, Rab3D, ABCB5, C-kit, K15, Ki67, and P63. Human lacrimal gland cells could form spheroids in vitro and then grow into mini-gland-like structures. PCR results showed proliferation and differentiation capability of those cultured cells.ConclusionsHuman lacrimal gland tissues contain precursor marker-positive cells and marker expression also was detected in ex vivo cultured cells, which showed differentiation capability.Translational RelevanceFuture studies of differentiation in human lacrimal gland tissue may aid in developing stem cell-based therapies for dry eye disease.
Highlights
The lacrimal gland is a tubuloacinar gland that is critical for ocular health and vision quality
The primary part of lacrimal gland is located within the upper temporal orbit, sending its secretions into an anastomosed duct system that delivers the tear component to the ocular surface
The fresh gland was collected in fetal calf serum (FCS)–rich Dulbecco’s modified eagle medium (DMEM) supplemented with antibiotic-antimycotic (Thermo Fisher Scientific, Waltham, MA) in 48C and transported to the lab, where it was immediately taken for processing
Summary
Human lacrimal gland samples were recovered by SightLife (Seattle, WA) within 24 hours postmortem. The fresh gland was collected in fetal calf serum (FCS)–rich Dulbecco’s modified eagle medium (DMEM) supplemented with antibiotic-antimycotic (Thermo Fisher Scientific, Waltham, MA) in 48C and transported to the lab, where it was immediately taken for processing. Cultured lacrimal gland cells were fixed with 4% paraformaldehyde (Millipore Sigma). To determine whether lacrimal gland progenitor cells underwent acinar differentiation, immunostaining for lactoferrin (Abcam, Cambridge, UK) and RAB3B (Santa Cruz Biotechnology) were performed. After appropriate washing with TPBS three times, the cells were incubated for 1 hour at room temperature with fluorescent dyes Alexa Fluor (AF) 488- or AF568-conjugated secondary antibodies (Thermo Fisher Scientific). Results were expressed as means 6 standard error of the mean (SEM) and were statistically compared by analysis of variance (ANOVA); P , 0.05 was considered statistically significant
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