Yumeng Zhang

Oil-producing sebaceous glands (SGs), attached to the upper/middle portion of hair follicles, are indispensable for maintaining skin hydration, and their dysfunction leads to dry skin. However, the molecular mechanisms underlying regulation of SG stem cells remain largely unknown. We identified transcription factor KROX20 as a marker of SG stem cells that sustains their stemness throughout SG morphogenesis and homeostasis. We developed an inducible mouse model in which ablation of KROX20-positive cells causes SG loss and rapidly and robustly induces dry, flaky, alopecia symptoms following induction. This model, termed xeroflacia (xero = xerosis, fla = flaky, cia = alopecia), provides a tool for studying the biology of dry skin. Furthermore, we found that KROX20 directly regulates Notch1 transcription to orchestrate the balance between SG stem cell self-renewal and differentiation. Small molecule drug modulation of Notch1 signaling to activate or inhibit the pathway enabled us to regulate SG differentiation to maintain skin oil levels, providing a proof of principle that other signaling pathways downstream of Krox20 could be therapeutic targets for SG-related diseases.

Meibomian gland dysfunction (MGD) is the leading cause of Dry Eye Disease (DED), accounting for approximately 90% of DED cases worldwide. The transcription factor KROX20 has been shown to mark stem cells that play a critical role in Meibomian gland (MG) development and homeostasis, however, the molecular mechanisms underlying these processes are not well understood. In this report, we used multiple Krox20 lineage tracing and ablation studies to investigate lineage commitment during MG morphogenesis and homeostasis. KROX20 marks MG stem cell pools, and regulates cellular contributions to the glands, as loss of Krox20 leads to MG atrophy, while its overexpression results in MG enlargement. We also found that Notch1 signaling is regulated by KROX20 and that ablation of the Notch1 gene in KROX20-expressing cells leads to MG atrophy, while Notch1 overexpression partially rescues MG atrophy caused by the loss of Krox20. Together, these results reveal a critical Krox20-Notch1 regulatory pathway in MG stem cells. Activation of Notch1 signaling may offer a novel approach to preventing or treating DED caused by depletion of the MG stem cell pool that can occur with ageing.

Mammalian skin is a vital barrier with the epidermis serving as its protective outer layer, continually undergoing renewal. Given that loss of the epidermis or its barrier function is lethal for mammals, multiple stem cell populations likely exist for the interfollicular epidermis (IFE), enhancing evolutionary survival. Here, we demonstrate that transcription factor KROX20 marks a heterogeneous stem cell population in the upper and middle mouse hair follicle (HF), partially overlapping with known HF stem cell markers in those regions. Lineage tracing in mice using different reporter lines shows that Krox20 -lineage cells migrate from the HF to the IFE, contributing to both basal and suprabasal layers during adulthood. Spatial transcriptomics data corroborate our findings. Depletion of epithelial Krox20 -expressing cells leads to epidermal hyperplasia and a disruption of stratification during homeostasis. Our study highlights the contribution of hair follicle Krox20 -lineage cells to the IFE and the regulation of epidermal homeostasis.

Epidermal stem cells control homeostasis and regeneration of skin and hair. In the hair follicle (HF) bulge of mammals, populations of slow-cycling stem cells regenerate the HF during cyclical rounds of anagen (growth), catagen (regression), and telogen (quiescence). Multipotent epidermal cells are also present in the HF above the bulge area, contributing to the formation and maintenance of sebaceous gland and upper and middle portions of the HF. Here, we report that the transcription factor KROX20 is enriched in an epidermal stem cell population located in the upper/middle HF. Expression analyses and lineage tracing using inducible Krox20-CreERT showed that Krox20 -lineage cells migrate out of this HF region and contribute to the formation of the bulge in the HF, serving as ancestors of bulge stem cells. In vivo depletion of these cells arrests HF morphogenesis. This study identifies a marker for an epidermal stem cell population that is indispensable for hair homeostasis.

Mesenchymal stem/Stromal cells (MSCs) have great therapeutic potentials, and they have been isolated from various tissues and organs including definitive endoderm (DE) organs, such as the lung, liver and intestine. MSCs have been induced from human pluripotent stem cells (hPSCs) through multiple embryonic lineages, including the mesoderm, neural crest, and extraembryonic cells. However, it remains unclear whether hPSCs could give rise to MSCs in vitro through the endodermal lineage. Here, we report that hPSC-derived, SOX17⁺ definitive endoderm progenitors can further differentiate to cells expressing classic MSC markers, which we name definitive endoderm-derived MSCs (DE-MSCs). Single cell RNA sequencing demonstrates the stepwise emergence of DE-MSCs, while endoderm-specific gene expression can be elevated by signaling modulation. DE-MSCs display multipotency and immunomodulatory activity in vitro and possess therapeutic effects in a mouse ulcerative colitis model. This study reveals that, in addition to the other germ layers, the definitive endoderm can also contribute to MSCs and DE-MSCs could be a cell source for regenerative medicine.