Research, as from Parfitt, et.al., seems to show that the terminal ends of the ductal epithelium are potential sites of adult progenitors or stem cells of the meibomian gland. There should be a stem cell reservoir within each meibomian gland to provide long-term self-renewal, but I hypothesize that drugs like Accutane/Isotretinoin destroy these reservoirs or make them malfunction and decreased blink rates decrease the necessary stimulation to these stem cells to keep producine holocrine cells full of meibum. 

I also hypothesize that there are 2 ways to destroy stem cells: Top-Down and Down-Up:
1. Top-Down: If the orifice of the meibomian gland is closed off from inflammation/demodex/ rosacea/poor make-up removal, etc., the back up of meibum will down-regulate the stem cell turnover and lead to their eventual atrophy. 
2. Down-Up: If the stem cell reservoir is directly destroyed by a drug/chemo/radiation/autoimmune disease, it will atrophy from the bottom to the top. 


We can see this in some meibographies where the residual meibum that lights up is at at the orifice of the gland (Down Up destruction) or at the base of the meibomian gland (Top-Down destruction). Sometiems the glands seems to have a hollow center where there is something causing scar tissue in the center and it looks almost moth eaten. 


In some patients who have many risk factors, there can be a more dramatic effect where a whole set of glands can disappear within months as seen on meibography. I have seen this in one patient who has an autoimmune disease and was on Accutane as a child. I hope to show this in my ASCRS presentation in DC in April. 





SLC


See notes below.
Thank you to the authors for a very helpful paper. 




From this, we hypothesize that holocrine secretion and turnover of the meibomian gland epithelia is directed from quiescent LRCs located at the junction between ductal and acinar tissue (Figure 7). As these cells are typically found as clusters with varied levels of label retention, a hierarchical model of cell turnover in the meibomian gland can be considered whereby the slowest cycling LRC may represent the meibomian gland stem cell. However, there remains the possibility that meibocyte progenitors may exhibit neutral competition and divide stochastically to regenerate each acinus, as is the case for Lgr5 intestinal stem cells (Snippert et al., 2010).

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Results

Short- and Long-Term Doxycycline Chase of the H2B-GFP/K5tTA Mouse to Characterize Label Dilution and LRCs in the Meibomian Gland

Prior to quantifying LRCs over long-term (26- to 56-day) chase, we evaluated the short-term (<16-day) proliferation status of the meibomian gland over the course of a 16-day chase in 10-μm cryosections (Figure 1). At postnatal day 28 following continued labeling from postnatal day 0, all epithelial cells in the meibomian gland exhibited nuclear GFP (Figure 1A), as demonstrated by a consistent fluorescence intensity across the gland in a pixel heatmap (Figure 1E). After 9-day doxycycline chase there was considerable variation in label dilution and cell-cycle rate between acini and the central duct, as some basal acinar cells were observed to be diluted of GFP (Figures 1B and 1F, red arrowheads). At 13-day doxycycline chase (Figures 1C and 1G), some acinar cells were completely diluted (red arrowhead) while others were only partially diluted (white arrowhead), implying that acinar turnover is not uniform, or synchronous across the meibomian gland. Moreover, the ductal epithelium remained largely GFP+ at the 13- and 16-day time points, while the majority of acini were diluted of GFP signal at 16 days post doxycycline (Figures 1D and 1H). This short-term chase experiment illustrates the difference in cell-turnover rates between each acinus, and between the acini and ductal epithelium of the meibomian gland.

Figure 1

Short-Term Doxycycline Chase of the H2B-GFP/K5tTA Mouse to Visualize Label Dilution in the Fastest Cycling Epithelial Cells of the Eyelid Meibomian Glands

From the 3D reconstructions generated by immunofluorescence tomography, a low quantity of slow-cycling, putative adult stem cells were localized in whole mouse meibomian glands by pulse-chase labeling of LRCs (Figure 2). At 9 days post doxycycline, the duct and the majority of acini remain strongly labeled with GFP and label dilution appears unevenly distributed across the gland (Figure 2A). After 28-day chase, acini are significantly diluted of GFP fluorescence with the exception of a small subpopulation of LRCs confined to the terminal regions of the ductal epithelium within an acinus (Figures 2B, 2E, and 2F). While LRCs are located at the terminal ends of the ductal epithelium at the entrance to an acinus, not all acini contain detectable LRCs by 42-day doxycycline chase, as observed in the 3D reconstructions of the whole meibomian gland (Figures 2C and 2G). The frequency of LRCs decreases from 28-day to 56-day chase (Figures 2D and 2H), and their decreasing number over time suggests that they remain functionally active while cycling at a remarkably slow rate.

Figure 2

Immunofluorescent Tomography 3D Reconstructions of the H2B-GFP/K5tTA Mouse Meibomian Gland after Pulse-Chase

After 3D reconstruction of DAPI and GFP signals in H2B-GFP/K5tTA mouse eyelids, we performed sequential immunostaining at 56-day doxycycline chase to characterize LRC differentiation and their proximity to K6+ ductal epithelium (Figure 3). All GFP+ slow-cycling cells were negative for the meibocyte differentiation marker PPAR-γ, confirming their undifferentiated state at 56-day chase (Figure 3A, yellow arrowhead). K6+ staining confirmed that LRCs are localized to the terminal ends of the ductal epithelium, or the ductule leading to the acinar epithelium (Figure 3B). LRCs maintain K5 expression, like all meibomian gland epithelia (Figure 3C); however, K6 expression varied in LRCs (Figures 3E–3H), suggesting there may be two subpopulations of LRCs. To characterize K6 expression in LRCs, we quantified the K6+ and K6− LRCs in the meibomian gland at 28-day (GFP+/K6− = 20 ± 3; GFP+/K6+ = 6 ± 1), 42-day (GFP+/K6− = 11 ± 1; GFP+/K6+ = 2 ± 0), and 56-day (GFP+/K6− = 9 ± 2; GFP+/K6+ = 2 ± 0) doxycycline chase and found that the K6− LRC subpopulation retains label stronger than the K6+ LRCs over longer chase time points.

Figure 3

Immunohistochemistry Staining of the H2B-GFP/K5tTA Mouse Meibomian Gland LRCs at 56-Day Doxycycline Chase

LRC Fluorescence Intensity Variation and Keratin 14-Confetti Lineage Tracing of Epithelial Progenitors

Based on their average GFP fluorescence intensity in the meibomian gland of experimental H2B-GFP/K5tTA mice, relatively GFP-weak LRC populations were the majority in the meibomian gland (Figures 4A and 4B). We calculated that there was an average of 1 ± 1 LRC+++, 3 ± 1 LRC++, and 10 ± 2 LRC+ subpopulations in the meibomian glands quantified at 42-day chase (n = 3). Vibrissae hair follicle LRC intensities at 42-day doxycycline chase (n = 3) were quantified as 18 ± 3 LRC+++, 29 ± 5 LRC++, and 37 ± 3 LRC+ subpopulations. Therefore, meibomian gland LRCs are considerably faster cycling than the hair follicle LRCs (Figures 4C and 4D), which retain label more strongly and are greater in number at 42-day doxycycline chase. This variation in label dilution correlates with a variation in cell-cycle rate among LRCs and may point to a hierarchical mode of cell division in the meibomian gland.

Figure 4

High-Resolution 3D Reconstructions of the Meibomian Gland and Vibrissae Hair Follicle Reveal a Gradient of Label Retention and GFP Fluorescence Intensity in LRCs at 42-Day Doxycycline Chase

We then used the K14CreERT2-Confetti reporter line to evaluate lineage tracing of K14 epithelial progenitors and determined that individual acini are likely to arise from single progenitors separate from the precursor cells of ductal epithelium (Figure 5). This was confirmed in the 2-μm-thick butyl methyl methacrylate (BMMA) sections, where confetti-labeled meibomian gland acini were frequently monochromatic in color or not labeled (Figure 5A). The evidence for unipotent acini progenitors was also supported when K6 immunostaining was performed on a confetti-labeled acini and the duct was completely distinct from YFP labeling in the K14CreERT2-Confetti mouse after a 23-week chase (Figure 5B). The labeling of entire acini in monochrome was analogous to the lineage-tracing pattern of K14 cells in the sebaceous gland, implying that each acinus in the gland arises from a single progenitor (Figure 5C). Fluorescence microscopy imaging of a 200-μm-thick vibratome section (Figure 5D) and 10-μm-thick cryosections (Figure 5E) showed that the ductal epithelium comprised multiple cell lineages, as contrasted by the monochromatic labeling of acini and the separate lineages of acini and duct at 23 weeks post tamoxifen injection in the K14CreERT2-Confetti mouse.

Figure 5

Lineage Tracing of K14+ Epithelial Progenitors in the K14CreERT2-Confetti Transgenic Mouse Meibomian Gland at 23 and 59 Weeks Post Tamoxifen Injection of a 10-Week-Old Mouse

At 59 weeks post tamoxifen injection of the K14CreERT2-Confetti mouse, monochromatic and dual-labeled acini were also observed in the meibomian glands (Figure 6A). This further implies that individual acini originate from a single progenitor when two fluorescent proteins (RFP and GFP) are stochastically expressed and retained long term, 59 weeks in this case. The 3D reconstruction generated by immunofluorescence tomography of the K14CreERT2-Confetti mouse meibomian gland (Figure 6B) also suggests that individual acini are derived from progenitor cells that renew the entire acini (Figure 6C), while the ductal epithelium is derived from multiple lineages separate from the acini (Figure 6D).

Figure 6

High-resolution 3D Reconstruction of the K14CreERT2-Confetti Mouse Eyelid after 59 Weeks Post Tamoxifen

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Discussion

In this study, we characterized the epithelial cell-cycle differences across the murine meibomian gland, as well as the LRC subpopulations that we propose to be the progenitors responsible for the renewal of the meibomian gland. The variation in cell-cycle rate that we found between each acinus, and between the acini and duct epithelium, suggests that they have distinct lineages that may operate on hierarchical cell-cycle programs. We used a lineage-tracing model to devise a hypothesis for cell turnover in the holocrine meibomian gland, in which unipotent LRCs renew individual acini and the ductal epithelium is derived from multiple, separate progenitor cells. In this way, the cell-cycle differences we observed between acini, and between acini and ductal epithelium, can be explained.

In the meibomian gland, low numbers of LRCs were observed after 28, 42, and 56 days of chase, which fulfill the slow-cycling, undifferentiated, and low population criteria of adult epithelial stem cells characterized in other tissues (Alonso and Fuchs, 2003, Stepp and Zieske, 2005, Tumbar et al., 2004). PPAR-γ immunostaining confirmed that the meibomian gland GFP+ LRCs are undifferentiated and do not synthesize lipid, whereas differentiated meibocytes undergoing lipogenesis enlarge and express nuclear PPAR-γ. The finding that meibomian gland LRCs retain label weaker and show greater reduction in numbers compared with the majority of hair follicle LRCs suggests that meibomian gland LRCs exist in lower populations and cycle faster than hair follicle LRCs in general. From this, we hypothesize that holocrine secretion and turnover of the meibomian gland epithelia is directed from quiescent LRCs located at the junction between ductal and acinar tissue (Figure 7). As these cells are typically found as clusters with varied levels of label retention, a hierarchical model of cell turnover in the meibomian gland can be considered whereby the slowest cycling LRC may represent the meibomian gland stem cell. However, there remains the possibility that meibocyte progenitors may exhibit neutral competition and divide stochastically to regenerate each acinus, as is the case for Lgr5 intestinal stem cells (Snippert et al., 2010).

Figure 7

A New Hypothesis for Holocrine Turnover of the Meibomian Gland Maintained by Quiescent Progenitors Located at the Terminal Ends of the Ductal Epithelium

Because of developmental similarities with skin appendages, and because they both secrete their contents in a holocrine manner, the meibomian gland is considered to be an enlarged, modified sebaceous gland and the mechanism of stem cell renewal is thought to show similar characteristics in these tissues (Knop et al., 2011). Epstein and Epstein (1966) originally showed that skin sebocytes differentiate over two distinct periods, i.e., between 7–9 days and 14–28 days, suggesting that migration and division of cells is heterogeneous in the peripheral basal layer (Epstein and Epstein, 1966), a finding supported by two distinct populations of sebocytes in the human forehead with different labeling indices (Plewig et al., 1971a, Plewig et al., 1971b). Our short-term chase (<16 days) experiments in the H2B-GFP/K5tTA mouse meibomian gland revealed a variation in cell turnover between acini themselves, and between acini and ductal epithelium, suggesting that acini are not in synchrony across a single meibomian gland. In the K14CreERT2-Confetti mouse, lineage tracing suggests that meibomian and sebaceous gland acini are derived from single-cell populations. When labeled, these acini are entirely composed of either one or two combined colors, which implies that renewal of an acinus comes from a single K14+ progenitor cell. Lineage tracing in the 3D reconstructions also verified that transgenic fluorescent labels are limited to individual acini and do not label multiple acini or the ductal epithelium.

In the 3D reconstructions generated by immunofluorescence tomography of H2B-GFP/K5tTA mice at each doxycycline chase time point, quiescent LRCs were localized to the ductule region where K6+ ductal epithelial cells terminate and the acini extend laterally. LRCs were observed to infrequently express K6, which indicates that two distinct unipotent LRC populations exist according to K6 expression. A unipotent progenitor giving rise to each acinus was supported by the lineage-tracing data, which show that acini are derived from a single progenitor cell over 59 weeks of long-term chase. If meibomian gland progenitors were bipotent, confetti labeling would extend from multiple acini into the ductal epithelium as a single progenitor renews both compartments of the gland, which was not observed herein. If LRCs are bipotent, the K6− LRC population is more likely to represent the adult stem cell population of the meibomian acinar population, as they are more label retaining than the K6+ population after long-term pulse-chase. How these LRCs remain functional yet quiescent, and whether an extracellular stem cell niche exists underlying the meibomian gland basal layer that controls their quiescence and activation, still remains unclear.

Our data suggest that the region where the duct transitions into an acinus harbors the meibomian gland progenitors, and localized hyperproliferation previously reported in this region (Parfitt et al., 2013) could constitute a transiently amplifying cell population. From this, we propose that LRCs are unipotent, much like the limbal epithelial stem cells that exclusively renew cornea (Amitai-Lange et al., 2015, Collinson et al., 2002, Di Girolamo et al., 2015, Dora et al., 2015), and most likely exist in two populations that separately renew acinar or K6+ ductal tissue. The heterogeneous nature of basal acinar cell divisions found previously (Epstein and Epstein, 1966, Plewig et al., 1971a, Plewig et al., 1971b) may relate to LRCs in these terminal regions of the ductal epithelium that went undetectable in these studies. Finally, senescence of these LRC progenitors with age may be responsible for the meibomian gland atrophy and dry eye disease that are so prevalent with aging (Jester et al., 2011, Nien et al., 2009, Nien et al., 2011, Parfitt et al., 2013).

In conclusion, the meibomian gland contains a low population of slow-cycling epithelial cells that may constitute an adult stem cell population continually renewing the holocrine tissue. These LRCs vary in cell-cycle length according to label-retention differences in the H2B-GFP/K5tTA mouse after pulse-chase. Interestingly, acini within the same gland were found to dilute label at different rates over a short-term chase, which suggests that their cycle is independent. Gene-expression studies of these cells will provide more targets for further identification and isolation of LRCs, as well as insights into the gene regulation of LRC quiescence. However, transcriptional profiling of meibomian gland LRCs remains challenging because of their proximity to the hair follicle LRCs. In the future, the isolation of human meibomian gland adult stem cells could provide new regenerative therapies and fundamental insights into holocrine systems that are so vulnerable to age-related atrophy, particularly in dry eye disease.