As tongue malignancy is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue malignancy, is unquestionably important. using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine. lineage tracing, has been applied for the identification of LESCs. In this review, we expose and discuss current progress in the identification of LESCs. To identify tissue-specific stem cells in the adult, a primary culture system that can reproduce the physiological environment and allow the differentiation of stem cells into various kinds of mature cells needs to be established. Using this system, we can precisely examine the pluripotency and the growth factor requirements of the stem cells. Recently, a three-dimensional (3D) organoid culture technique using extracellular matrix has been developed for the small intestine , belly , and colon . This technique allows the generation of organoids made up of multilayered epithelial structures from crypts and even from single stem cells isolated TMP 269 from adult animals. In this review, we expose a new lingual epithelial organoid culture system as well as early lingual epithelial cell culture systems. 2. Lingual Stem Cell Markers 2.1. Keratin 5 and Keratin 14 Keratin 5 (K5) and keratin 14 (K14), intermediate filament proteins, are known to be expressed in basal keratinocytes of stratified epithelium in the skin, and the mutation or absence of both proteins makes the cellular architecture in basal keratinocytes vulnerable . Similar hCIT529I10 to the skin, immunohistochemistry analyses of mouse tongue revealed that both proteins are expressed at the highest level in the basal layer of the tongue epithelium. The expression level decreases in each layer closer to the surface epithelial layer [7,8] (Table 1). Luo reported that K5-positive lingual epithelial cells (LECs) obtained from K5-eGFP mice could generate a multilayered squamous keratinized epithelium when these cells were cultured on a collagen-fibroblastic cell-matrix in the presence of epidermal growth factor (EGF) and fibroblast growth factor 7 (FGF7) , supporting that K5-positive cells include lingual stem cells and/or progenitors. Table 1 Markers of lingual epithelial stem cells (LESCs) and the results of their lineage tracing experiments. lineage tracing assay with mice, Okubo found that NTPDase2 colocalized with the glial glutamate/aspartate transporter (GLAST), which is regarded as a marker of type I cells in taste buds, by using immunohistochemical and enzyme histochemical staining methods . In contrast, Li exhibited that LECs in basal and suprabasal cell layers as well as taste bud cells in fungiform and circumvallate papillae express NTPDase2 by using hybridization with an NTPDase2 probe  (Table 1). Moreover, a genetic tracing study of NTPDase2-positive cells (doxycycline inducible, NTPDase2-rtTA/TeTO-Cre; RosaLacZ reporter system) revealed that descendant cells derived from the NTPDase2-positive cells generated filiform, fungiform, and circumvallate papillae as well as taste bud cells in fungiform papillae and circumvallate papillae. From the results, they propose the presence of common progenitor cells that contribute to both taste bud cells and LECs. However, by the single-color lineage tracing method using the Rosa26 reporter mouse in this study, the TMP 269 proof for the bipotency of K14 positive lingual stem/progenitor cells was not sufficient, because TMP 269 the different clones next to each other could show the same color. 2.3. Multicolor Lineage Tracing Method To precisely examine the fate of each stem cell, the multicolor lineage tracing method is now considered as one of the most powerful techniques. The multicolor lineage tracing method was originally developed to analyze TMP 269 lineage associations between blood and endothelial cells within yolk sac blood islands of mice . However, in the original method, multicolor chimeras were generated by injecting.