The infrequently dividing or so-called slow-cycling nature of cells within tissues has been linked to stemness in several adult tissues including mouse skin. Slow-cycling cells defined as H2B-GFP or DNA label retaining cells (LRCs) are thought to reside at the top of the lineage hierarchy, as stem cells, giving rise to actively-cyclig cells (non-LRCs), which may function as transit-amplifying (TA) short-lived progenitor cells. Despite the crucial importance of the epidermis for essential body functions and for skin regeneration therapy, molecular characterization of stem and TA cells within the basal layer of the inter-follicular epidermis has not been achieved. Consequently, we lack the specific markers to accurately define the dynamic stem cell behavior within this tissue and to understand the regulatory factors maintaining stemness. In our preliminary study, we have isolated LRCs and non-LRCs from the basal layer of the epidermis and analyzed the molecular signature of these cells by microarray. Here we propose to characterize the expression pattern of several putative epidermal stem and TA cell markers identified in our microarray, including one novel cell surface marker for basal layer LRCs. We will analyze the lineage potential of subpopulations of basal layer cells by three functional assays: colony formation activity, regeneration potential in cell transplantation, and lineage tracing. These data will reveal long-term stem cell potential and lineage hierarchy of epidermal basal cell subpopulations in vitro and in vivo and will provide new tools for studying stem cell regulation in the epidermis. Data obtained from our proposed experiments will have global relevance for stem cell-based therapy of human skin injury or skin replacement, where slow-cycling cells are thought to play central roles.
Tissue stem cells reside in regenerative tissues such as skin, and are responsible for supplying cells to maintain the normal tissue function and to repair injury throughout life. Recent evidence, although controversial, has challenged the model that proposes stem cells divide infrequently to protect their genome from accumulating errors. We have developed tools to isolate 'dormant' and 'active' cells from the skin epidermis and characterize their biochemical properties, which will allow us to examine the pathways that maintain these cells throughout life. Work in the mouse system should translate to human skin, and will provide a theoretical basis for understanding the molecular basis of disease, when stem cells become de-regulated. A hierarchical stem/transit-amplifying (TA) cell model predicts that stem cells (SCs) divide more infrequently than their immediate daughter cells, which represent a short-lived, more differentiated population of cells called TA cells. The slow-cycling nature of SCs is thought to be a mechanism of preventing genetic mutations, tumorigenesis and aging caused by repeated, life-long cell divisions. However, this prevailing view has been recently challenged, since actively-cycling cells in mouse intestine were found to act as SCs (1). Adult mouse skin is organized into inter-follicular epidermis and associated appendages, including hair follicles and sebaceous glands. The epidermis has SCs in the basal layer (BL), which proliferate and provide differentiated cells in the upper, suprabasal layers (sBL). Recently, a study using single cell lineage tracing and mathematical modeling suggested that epidermal BL contains both slow-cycling stem cells and active-cycling TA cells (2). However, due to lack of specific molecular markers for the BL subpopulations, lineage relationship and regulatory factors of these cells remain elusive. We employed our previously developed tet-off transgenic mice to mark slow-cycling cells in skin epithelium as histone H2BGFP label-retaining cells (LRCs) (3). We further combined this system with an established Cre- loxP mouse system to specifically label epidermal BL and sBL cells and exclude the rest of skin compartments. We have isolated and analyzed LRC and non-LRCs from the basal and supra-basal epidermal cell populations by microarray. We found hundreds of genes, not related to cell cycle, which were differentially expressed in LRCs and non-LRCs in the BL of the epidermis, which demonstrated that they are molecularly distinct. We hypothesize that slow- and actively-cycling cells in the epidermal BL may also have a distinct nature in their stem cell potential, cellular fate and regulatory mechanisms. To address this hypothesis, we will identify specific bio-markers for experimental testing of the long-term self-renewal/differentiation potential and lineage hierarchy of slow- and actively-cycling cells in the mouse epidermis. Aim 1. To characterize subpopulations of BL cells separated based on LRC-marker ILR expression. Using microarray analysis of the sorted epidermal cells, we found that LRCs and non-LRCs in the epidermal BL differentially express several cell surface markers that would permit separation of the 2 BL subpopulations. We confirmed by fluorescence activated cell sorting that the interleukin receptor (ILR) is expressed in ~30% of BL epidermal cells from wild type mice, defined as ?6+/Sca1+ cells. We will test stem cell characteristics in the ILR+ and ILR- BL subpopulations by long-term colony-forming assay in vitro and cell transplantation in vivo. This will provide the means to separate BL epidermal sub-fractions from the mouse skin, as more defined SC populations, which will prove useful in future functional studies that model human disease in mutant mice. Aim 2. To validate putative epidermal LRC and non-LRC markers by lineage-tracing in vivo. Among the genes expressed differentially by BL LRCs and non-LRCs in our microarray we found several candidates, for which mice carrying a knock-in CreER allele are already available. We will validate the expression patterns of these genes in different mouse genetic backgrounds by immunostaining and mRNA in situ hybridization. For a select number of candidates we will test long-term stem cell potential of BL LRCs and non-LRCs by in vivo lineage tracing. These experiments will address a possible functional heterogeneity of slow- and actively- cycling cells within epidermal BL. Moreover, they will provide specific markers for direct targeting of these subpopulations and for understanding specific molecular mechanisms of epidermal SC homeostasis. Our work will reveal the cellular potential and in the future will provide tools to analyze the molecular control of subpopulations of slow- and actively-cycling cells, and ultimately of stem cells, in the mouse epidermis. The cell fate mapping, transplantation and in vitro colony-forming assay will provide evidence on how slow- and actively-cycling cells behave, interplay and contribute to tissue homeostasis and repair, which remain poorly understood to date. Some of the markers identified in our arrays have been previously reported expressed in human epidermis, pointing to the translational value of our work. Our data will provide the missing basic science platform for modeling human skin disease by using tractable genetic model systems that can interrogate more directly the role and regulation of of slow-cycling SCs. In addition our work will have relevance for better isolation and manipulation of human epidermal stem cells for future use in skin replacement.
| Wang, Alex B; Zhang, Ying V; Tumbar, Tudorita (2017) Gata6 promotes hair follicle progenitor cell renewal by genome maintenance during proliferation. EMBO J 36:61-78 |
| Sada, Aiko; Jain, Prachi; Wang, Sherry et al. (2017) Slc1a3-CreER as a Targeting Tool for the K6+ Epithelial Stem Cell Niche and its Precursors during Mouse Hair Follicle Cycle. J Invest Dermatol 137:1569-1571 |
| Sada, Aiko; Jacob, Fadi; Leung, Eva et al. (2016) Defining the cellular lineage hierarchy in the interfollicular epidermis of adult skin. Nat Cell Biol 18:619-31 |
