Biomarkers for Progenitor and Differentiated Epithelial Cells in the Human Meibomian Gland

Abstract

The meibomian gland (MG) is a sebaceous gland that secretes through a holocrine process. Because such secretion requires the destruction of MG acinar epithelial cells, they need constant renewal and differentiation. The processes that promote these regenerative events in the human MG are unknown, nor is it known how to distinguish MG progenitor and differentiated cells. We discovered that Lrig1 and DNase2 serve as biomarkers for human MG progenitor and differentiated cells, respectively. Lrig1 is expressed in MG basal epithelial cells in the acinar periphery, a location where progenitor cells originate in sebaceous glands. DNase2 is expressed in the differentiated epithelial cells of the MG central acinus. Furthermore, proliferation stimulates, and differentiation suppresses, Lrig1 expression in human MG epithelial cells. The opposite is true for DNase2 expression. Our biomarker identification may have significant value in clinical efforts to restore MG function and to regenerate MGs after disease-induced dropout. Stem Cells Translational Medicine 2018;7:887-892.

Keywords: Differentiation; Proliferation; Stem cell plasticity; Stem/progenitor cell.

Figure 1

Identification of K14, K6, and Lrig1 in the human meibomian gland (MG). (A): K14 is present in both ductal and acinar epithelial cells, whereas K6 expression is restricted to ductal cells. (B): Lrig1‐positive cells are located only in the basal layer of the MG acinus. Scale bar = 50 μM. Abbreviations: K6, cytokeratins 6; K14, cytokeratins 14.

Figure 2

Differential appearance of Lrig1 in proliferating versus differentiated human meibomian gland epithelial cells (HMGECs). In parallel with a change in cellular morphology (A), Lrig1 expression in proliferating HMGECs (B) (PM frame) appears to be lost when cells differentiate (B; DM and AZM frames). (C): This decrease in Lrig1 levels was confirmed by Western blot (n = 3 experiments, **p < .01). Scale bar = 50 μM. Abbreviations: AZM, azithromycin; DM, differentiation medium; PM, proliferation medium.

Figure 3

Stimulation of Lrig1 expression in immortalized human meibomian gland epithelial cells. (A) Cells were cultured in basal KSFM or PM media. The latter promoted both Lrig1 and PCNA appearance, as shown by Western blot (n = 3 experiments, *p < .05) (B). Abbreviations: KSFM, keratinocyte serum‐free medium; PCNA, proliferating cell nuclear antigen; PM, proliferation medium.

Figure 4

Cellular location of DNase2 in the human meibomian gland (MG). To identify neutral lipids and lysosomes, cells were stained with LipidTOX 19 and antibodies to LAMP‐1 31. LipidTOX staining of neutral lipids is present in the epithelial cells of the acinus, but not the basal layer (A). DNase2 staining localizes in central cells of the acinus (B). DNase2 is located in neutral‐lipid (D) containing lysosomes (C) in the human MG. Note the cytoplasmic lipid droplets (D), which are also found in sebocytes 32. Scale bar = 50 μM. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; LAMP‐1, lysosomal‐associated membrane protein 1.

Figure 5

Differential expression of DNase2 in proliferating versus differentiated human meibomian gland epithelial cells (HMGECs). DNase2 is manifest in HMGECs when cultured in DM or AZM, but not in PM, as shown by immunofluorescence (A) and Western blot (B) (n = 3 experiments,**p < .01). Scale bar = 50 μM. Abbreviations: AZM, azithromycin; DM, differentiation medium; PM, proliferation medium.

Figure 6

Plasticity of biomarkers in human meibomian gland epithelial cells (HMGECs). Switching the culture media from PM to DM to PM (A, B), or from DM to PM to DM (C, D) induces corresponding shifts in cellular morphology (A, C) and Lrig1 and DNase2 expression (B, D). Proliferation stimulates, and differentiation reduces, Lrig1 appearance in HMGECs (A, B). The opposite response is found for DNase2 (C, D). Scale bar = 100 μM. Abbreviations: DM, differentiation medium; PM, proliferation medium.