Background

Adult stem cells play a key role in tissue homeostasis and regeneration because of their ability to self-renew and generate differentiated cells. Age-related changes in these stem cells are closely associated with aging and age-related tissue diseases [1,2]. Many studies have aimed to identify changes in adult stem cells related to aging and develop anti-aging agents to slow the aging process [3–8]. Although studies have shown that various factors contribute to aging, there are limitations to quantifying these factors. Currently, there is no statistical method for quantifying adult stem cell aging.

First, a suitable model system is required to quantitatively study adult stem cell aging. Drosophila, due to their short life span and genetically modifiable midgut function, are a widely utilized model organism for aging research, encompassing investigations of adult stem cells and their microenvironmental as well as changes associated with aging [2,8,9]. In the adult Drosophila midgut, intestinal stem cells (ISCs), marked by delta (Dl), are the only proliferation cells that give rise to two distinct differentiated progenies: enterocytes (ECs), which arise from enteroblasts (EBs) induced by strong Notch (N) signaling, and enteroendocrine (EE) cells, which are derived from EBs activated by weak N signaling [10]. These four types can be identified using specific markers such as Dl (ISCs), phospho-histone H3 (PH3, dividing ISCs), esg-GFP (ISCs and EBs), Su-GFP (EBs), Pdm and Myo-GFP (ECs), and Prospero (Pros) and Pros-GFP (EE cells) [7–8,10].

Second, utilizing appropriate markers is crucial for quantitatively assessing ISC aging. Using an anti-PH3 antibody, Choi et al. revealed age-related increases in Drosophila ISC division, which was corroborated by subsequent studies [3,11,12–16]. The anti-PH3 antibody specifically binds to phospho-histone H3 at serine 10, a modification that occurs during mitosis; therefore, it is widely used as a marker for identifying mitotic cells [14]. Using an anti-γH2AX antibody, Park et al. observed age-related increases in DNA damage accumulation in ISCs [17]. The anti-γH2AX antibody specifically recognizes and binds to phosphorylated histone H2AvD at serine 137, a modification analogous to the γH2AX phosphorylation in mammals [17]. Using an anti-γ-tubulin antibody, Park et al. revealed that age-related increases in abnormal centrosome amplification occur in ISCs and renal stem cells (RSCs) [14,15,18]. The anti-γ-tubulin antibody specifically recognizes and binds to γ-tubulin, a key protein in microtubule nucleation and organization in the division of eukaryotic cells [14,15].

Third, sufficient aging and age-related conditions are required to quantitatively study adult stem cell aging. It is well established that 60-day-old flies maintained at 25 °C and 45-day-old flies maintained at 29 °C serve as effective models for aging when compared to young flies [14–17]. In addition, the model of intrinsic oxidative stress Catalase heterozygous mutant flies is a widely used age-related model [14,18].

Fourth, appropriate anti-aging agents are essential for quantitatively assessing their effects on adult ISCs. Previous studies have reported that metformin, a commonly prescribed medication for the management of type 2 diabetes, and β-hydroxybutyrate, a type of ketone body produced during periods of fasting, prolonged exercise, or low-carbohydrate diets, exhibit anti-aging properties [15,19]. In recent studies, age-related declines in Vitamin D (VitD) synthesis and VitD receptor (VDR) expression have been associated with age-related diseases [16]. However, few studies have explored the role of VitD/VDR in adult stem cells, highlighting the need for further related investigations. In this study, by utilizing the age markers of Drosophila ISCs and age-related conditions, we aimed to provide a detailed and precise method for the quantitative measurement of adult stem cell aging. This method can be used for the quantitative analysis of anti-aging agent development and efficacy research, which will contribute to reducing research costs and shortening the research period spent in anti-aging fields in the future.