Epithelial stem cells in Meibomian gland development and homeostasis

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Project Summary Dry eye disease (DED), which occurs when there is inadequate lubrication of the eyes due to insufficient tears or excessive tear evaporation, affects over 16 million adults in the U.S., and the prevalence continues to increase. If left untreated, DED can lead to serious problems including inflammation of the eye, corneal abrasions, and even vision loss. While DED can be due to a variety of causes, ~90% of cases are due to Meibomian gland dysfunction (MGD). The Meibomian glands (MGs) are holocrine sebaceous glands located in the eyelids. Differentiated meibocytes within the MGs secrete meibum, a mixture of lipids and proteins that coats the ocular surface and prevents tear evaporation. MGD can be due to reduced meibum secretion, resulting from abnormal MG activity, blockage of the MG orifice due to hyper-keratinization, or structural defects such as MG atrophy or dropout. Another possible cause of MGD is stem cell exhaustion, in which MG resident stem cells are unable to replenish meibocytes due to dysregulated stem cell differentiation. MG development is well-conserved in mice and humans. In mice, MGs begin to develop at embryonic day 18.5 from the ectodermal epithelium; the MG anlage grows out of the eyelid mesenchyme, elongates, branches, and differentiates into ductules that form the acini; one MG contains clusters of these secretory acini which contain the meibocytes. MGs are fully formed by postnatal day 15. Throughout life, meibocytes are replenished from a stem cell population within the acini. While MG morphological development has been well described, the identity of the stem cell population(s) that gives rise to the MG remains controversial. Additionally, the resident stem cell population responsible for replenishing meibocytes after holocrine secretion in mature MG has not been definitively identified. Previous studies indicate there is a limited number of precursor meibocytes, which may be the cause of stem cell exhaustion that can occur with aging. Thus, the stem cell origin(s) and molecular mechanisms underlying MG development and homeostasis, i.e. replacement of acinar meibocytes throughout life, remain major knowledge gaps in the field. A recent study from our laboratory identified Krox20 as a marker of a stem cell population in the hair follicle that differentiates into hair shaft progenitor cells in the hair matrix, and ultimately constitutes the structural component of the hair shaft (Liao, G&D, 2017). Follow-up preliminary studies revealed that a population of Krox20+ cells also gives rise to the MG and depletion of these Krox20+ cells or the KROX20 protein results in lack of MGs. These data highlight a critical role for Krox20 in MG development. We hypothesize that Krox20 marks a novel stem cell population that gives rise to the MG. Our objectives in this proposal are to investigate the role of these Krox20+ cells in MG morphogenesis and homeostasis, and delineate the mechanisms by which KROX20 exerts its biological function in MG. The identification, isolation, and study of this previously unidentified MG stem cell will provide insights that will advance the field, filling a current knowledge gap, as well as providing a novel murine model of MGD-based DED that offers a platform for testing potential therapies for DED.