Dentistry, Faculty of
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International Journal of Molecular Sciences 26 (7): 3144 (2025)
Harris, Ashlee
Burnham, Kaylee
Pradhyumnan, Ram
Jaishankar, Arthi
Häkkinen, Lari
Góngora-Rosero, Rafael E.
Piazza, Yelena
Andl, Claudia D.
Andl, Thomas
The mechanisms that influence human longevity are complex and operate on cellular, tissue, and organismal levels. To better understand the tissue-level mechanisms, we compared the organization of cell proliferation, differentiation, and cytoprotective protein expression in the squamous epithelium of the esophagus between mammals with varying lifespans. Humans are the only species with a quiescent basal stem cell layer that is distinctly physically separated from parabasal transit-amplifying cells. In addition to these stark differences in the organization of proliferation, human squamous epithelial stem cells express DNA repair-related markers, such as MECP2 and XPC, which are absent or low in mouse basal cells. Furthermore, we investigated whether the transition from basal to suprabasal is different between species. In humans, the parabasal cells seem to originate from cells detaching from the basement membrane, and these can already begin to proliferate while delaminating. In most other species, delaminating cells have been rare or their proliferation rate is different from that of their human counterparts, indicating an alternative mode of how stem cells maintain the tissue. In humans, the combination of an elevated cytoprotective signature and novel tissue organization may enhance resistance to aging and prevent cancer. Our results point to enhanced cellular cytoprotection and a tissue architecture which separates stemness and proliferation. These are both potential factors contributing to the increased fitness of human squamous epithelia to support longevity by suppressing tumorigenesis. However, the organization of canine oral mucosa shows some similarities to that of human tissue and may provide a useful model to understand the relationship between tissue architecture, gene expression regulation, tumor suppression, and longevity.
Academic Editor: Nicola AlessioReceived: 17 February 2025Revised: 19 March 2025Accepted: 26 March 2025Published: 28 March 2025Citation: Harris, A.; Burnham, K.;Pradhyumnan, R.; Jaishankar, A.;Häkkinen, L.; Góngora-Rosero, R.E.;Piazza, Y.; Andl, C.D.; Andl, T.Human-Specific Organization ofProliferation and Stemness inSquamous Epithelia: A ComparativeStudy to Elucidate Differences in StemCell Organization. Int. J. Mol. Sci. 2025,26, 3144. https://doi.org/10.3390/ijms26073144Copyright: © 2025 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license(https://creativecommons.org/licenses/by/4.0/).ArticleHuman-Specific Organization of Proliferation and Stemness inSquamous Epithelia: A Comparative Study to ElucidateDifferences in Stem Cell OrganizationAshlee Harris 1, Kaylee Burnham 1, Ram Pradhyumnan 1, Arthi Jaishankar 1, Lari Häkkinen 2 ,Rafael E. Góngora-Rosero 1 , Yelena Piazza 3, Claudia D. Andl 1 and Thomas Andl 1,*1 Burnett School of Biomedical Sciences, University of Central Florida, Orlando, FL 32826, USA;burnhamkb@gmail.com (K.B.); ra370988@ucf.edu (R.P.); euraffa@gmail.com (R.E.G.-R.)2 Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia,Vancouver, BC V6T 1Z1, Canada; lhakkine@dentistry.ubc.ca3 College of Medicine, University of Central Florida, Orlando, FL 32827, USA* Correspondence: thomas.andl@ucf.eduAbstract: The mechanisms that influence human longevity are complex and operate oncellular, tissue, and organismal levels. To better understand the tissue-level mechanisms,we compared the organization of cell proliferation, differentiation, and cytoprotectiveprotein expression in the squamous epithelium of the esophagus between mammals withvarying lifespans. Humans are the only species with a quiescent basal stem cell layer thatis distinctly physically separated from parabasal transit-amplifying cells. In addition tothese stark differences in the organization of proliferation, human squamous epithelialstem cells express DNA repair-related markers, such as MECP2 and XPC, which are absentor low in mouse basal cells. Furthermore, we investigated whether the transition frombasal to suprabasal is different between species. In humans, the parabasal cells seem tooriginate from cells detaching from the basement membrane, and these can already beginto proliferate while delaminating. In most other species, delaminating cells have been rareor their proliferation rate is different from that of their human counterparts, indicating analternative mode of how stem cells maintain the tissue. In humans, the combination of anelevated cytoprotective signature and novel tissue organization may enhance resistance toaging and prevent cancer. Our results point to enhanced cellular cytoprotection and a tissuearchitecture which separates stemness and proliferation. These are both potential factorscontributing to the increased fitness of human squamous epithelia to support longevity bysuppressing tumorigenesis. However, the organization of canine oral mucosa shows somesimilarities to that of human tissue and may provide a useful model to understand therelationship between tissue architecture, gene expression regulation, tumor suppression,and longevity.Keywords: squamous epithelial biology; esophageal stem cells; spatial biology; multipleximmunofluorescence; comparative biology; MECP2; XPC1. IntroductionWithin the class of mammalia, few species exhibit a maximum lifespan above 100years [1]. Humans belong to this small group of mammals with extreme longevity. Themolecular basis of longevity in mammals remains poorly understood, but it is widelybelieved that all organs and tissues in long-lived organisms must be optimized, as even theweakest link can determine overall morbidity [2].Int. J. Mol. Sci. 2025, 26, 3144 https://doi.org/10.3390/ijms26073144Int. J. Mol. Sci. 2025, 26, 3144 2 of 26A contributor to morbidity is cancer, which primarily occurs in self-renewing epitheliaand is associated with stem cell proliferation rates [3]. Aging in such epithelia leads toloss of repair capacity, cellular senescence, and clonal selection of cell populations thatmay become malignant [4]. To maintain these epithelia and prevent them from becomingthe “weakest link” the epithelial stem cells likely have enhanced protection and increasedresilience programs [5].Different organs or tissues may employ distinct mechanisms to promote longevity,and each mammalian order—or even each mammalian species—may have evolved uniquestrategies to address the challenges of long life. For example, the liver cells of long-livedmammals display a coordinated reprogramming of gene expression at a genome-widescale, including reduced expression of genes involved in the metabolism of saturated andunsaturated fatty acids [6]. In contrast, bats, which are exceptionally long-living consideringtheir small size, exhibit adaptations at the proteome level. Comparative analyses of bat andmouse liver cells suggest that bats have evolved enhanced protein stability, resistance toprotein oxidation, and overall improved protein homeostasis [7].Longevity in mammals is linked to improved DNA repair, protein quality control,and immune function [8–12]. However, beyond enhancing repair and quality control, acritical protective mechanism involves avoiding processes that elevate the risk of muta-tions and cancer. Because DNA replication and mitosis are among the riskiest processesfor introducing mutations [3,13], fibroblasts from some small long-lived rodents exhibitmarkedly reduced growth rates compared to those from short-lived species [14]. That studyfurther indicated that strategies to achieve longevity vary significantly: large rodents tendto rely on replicative senescence, whereas small rodents modulate cell division rates as anadaptive mechanism [14].Organizing the germinative compartment of the epithelium into stem cells and transit-amplifying cells (also known as originally as transient amplifying cells [15] or transitoryproliferative cells [16]) can maximize both cell renewal and the protection of genetic in-tegrity [5,17]. This physical separation is seen in human squamous epithelia, such as thosefound in the oral cavity, esophagus, anus, vagina, and ectocervix, and is unique amongstudied mammalian species.To understand the potential role of this distinct organization in longevity, cancer-protection, and its fate in cancer development, we compared the expression of genesassociated with cellular protection against DNA damage in human squamous epithelialcells to that in other mammalian species. Using high-plex immunofluorescence stainingthat we validated for cross-species analysis and image segmentation in conjunction withsingle-cell RNA sequencing data, we analyzed the differences in squamous epithelialstem cell biology between humans and 15 other mammals (Supplementary Table S1).Our specific focus was to detect differences between short-lived and long-lived species,with emphasis on cellular protection mechanisms. The toolset included antibodies andmultiplexing that can characterize tissue architecture and cell types and adaptive changesin morphology and tissue architecture that may drive longevity (references and basicinformation on these proteins are provided in Supplementary Table S1). This approachis based on the observations outlined by Carroll [18] that alterations in gene expression,rather than DNA and protein sequence changes, drive adaptations including longevity [6].Thus, our approach interrogated both changes in protein expression levels and patterns aswell as tissue architecture.Such a detailed, two-pronged approach has led us to identify a quiescent basal cell layerin humans and define its gene expression pattern using small tissue samples. Two markers,XPC and MECP2, stood out. XPC is an essential component of the DNA damage controlmachinery and its loss in humans leads to dramatically reduced life expectancy [19–21]. Its roleInt. J. Mol. Sci. 2025, 26, 3144 3 of 26in squamous epithelial outside of the skin has not been explored in detail. A protein thatcan interact with XPC is MECP2 [19]. It has also become associated with the protectionof genomic integrity and its mutation results in reduced life expectancy in mice andmen [22,23]. We identified both as markers of quiescent stem cells of human squamousepithelia and show that they interact specifically in these cells. Our results indicate thathuman basal cells exhibit elevated expression of cytoprotective proteins, including XPCand MECP2, which is not observed to the same extent in the basal cells of other species.Moreover, this elevated expression is lost in precancerous and cancerous lesions, supportingthe hypothesis that MECP2/XPC expression and basal cell quiescence may play a tumor-suppressive role. However, dog squamous epithelia have some features of proliferationorganization that could make them useful models of the human condition and for oralcancer studies.2. Results2.1. Establish a Set of Antibodies Suitable for Cross-Species Analysis of Proliferationand “Stemness”To establish a multispecies multiplex immunofluorescence comparison, we evaluateda set of antibodies that define proliferation, differentiation, stemness, and cell outline insquamous epithelia. We initially tested 29 antibodies (Supplementary Table S1). In brief,proliferation marker antibodies such as PCNA, phosphor-serine 10 Histone H3 (pH3), andMCM2 worked nearly universally in the mammals we tested. To define epithelial cells andtheir outlines or cell bodies, we examined antibodies against CDH1 (E-cadherin), KRT14,CTNNB1 (beta-catenin), and CD44. With few exceptions, all these antibodies work well inmammals, but only the anti-CDH1 antibody also detected CDH1 in chicken.2.2. Basal-Suprabasal Cell Dichotomy in Humans and Absent in Other Analyzed Species: BasalCell Quiescence and Accompanying MECP2 and XPC Expression Are Human FeaturesWith a set of broadly applicable antibodies on hand, we determined quantitativeand qualitative differences in protein expression. MECP2 and XPC are novel markers ofthe basal cell layer, especially in humans, and their specific basal expression in certainmammals prompted us to further analyze them. They are also unique in the sense thatrelated proteins do not exhibit basal specific expression (Supplementary Figure S1) andXPC binding partners are expressed basally while other DNA damage related proteins aresuprabasally expressed (Supplementary Figure S2). Therefore, we hypothesized that theiranalysis may offer new insights into differences in epithelial organization in mammalsand how DNA damage repair is specifically managed in quiescent basal stem cells. Toput their expression into context, we also assessed expression of TP63 (general epithelialmarker with preferences to basal cell expression), Histone H1 (expressed in all cells andtherefore useful for normalization), and MCM2 (a reliable proliferation marker). Referencesfor protein functions and protein classification are found in Supplementary Table S1. BothMECP2 and XPC are barely expressed in mouse squamous epithelia (ca. 5 and 11-foldlower in mouse compared to human basal cells, respectively, Figure 1 and SupplementaryTable S2). As expected, proliferation was low in this MECP2high human basal cell layer (thequiescent stem cell layer), while it was high in MECP2− parabasal cells (Figure 1A). Onthe other hand, most other mammalian species completely lacked a parabasal proliferativepopulation and proliferation and stemness were restricted to the basal cell layer. The classicexample for this organization can be found in rodents, where expression of a proliferationmarker such as MCM2 is restricted to the basal cells (Figure 1D versus 1A, Figure 1H).Using a multiplex immunofluorescence staining technique (Supplementary Table S3) andmeasuring the fluorescent signal in each epithelial cell after manual cell segmentation,Int. J. Mol. Sci. 2025, 26, 3144 4 of 26we analyzed MECP2, XPC, P63 (markers associated with the basal cell layer), MCM2(proliferation marker) and histone H1 (normalizer) in esophageal and oral squamousepithelial cells (Figure 1). The differences in human epithelial organization are highlightedwhen comparing basal and suprabasal expression by measuring fluorescent intensities inindividual cells and normalizing expression either to stromal cells (Figure 1E–H), using histoneH1 (a marker expressed at similar levels in basal and suprabasal cells), or using unnormalizeddata (Supplementary Table S2). In humans, there is a pronounced and abrupt drop in expressionof MECP2 and XPC from basal to suprabasal cells (Supplementary Table S2, ca. 16- and 5-fold,respectively) and humans express about 5-fold more MECP2 and 11-fold more XPC in theirbasal cells than mice do (Supplementary Table S2). This drop from basal to suprabasalcell expression is still present although attenuated in monkey (Figure 1B,E,F), and absentin rodents such as rats and mice (Figure 1C–F). Interestingly, out of the species tested,dogs showed a surprising resemblance to humans, suggesting an even higher similaritybetween dog and human tissue organization than human and monkey. To make ouranalysis more robust and to control for differences between species and tissue quality, weused histone H1 to serve as a reference value to normalize gene expression (similar toapproaches used in qRT-PCR and Western blotting). All the 22 analyzed markers except forTP63 were also expressed in stromal cells. Therefore, we also used these stromal cells asinternal normalizers for the epithelial expression of MECP2, XPC, MCM2, and Histone H1.Thereby, we performed normalization based on a reference protein (histone H1) and basedon reference cells (stromal cells). The noted expression of the proteins in the stromal cellsalso suggests that low or absent expression of a marker in the epithelium in the samples isunlikely to be an artifact.In addition to MECP2 and XPC, we also investigated the keratinocyte marker TP63which shows generally the highest expression in basal cells (Supplementary Table S2).Normalization to histone H1 overall preserved this trend. Therefore, TP63 can be regardedas a squamous epithelial basal stem cell marker as it has been suggested in the literature [24].In contrast to the widely used animal models, mice and rats, which only proliferate inthe basal cell layer, we also included less conventional animal models such as pigs and dogsin our study to explore alternative models of squamous epithelial cell biology. Interestingly,the dog sample showed areas of basal cell quiescence reminiscent of a human epitheliumsuggesting that dogs may be suitable to explore characteristics of this cell layer in a waythat is also relevant for humans. This observation is in line with our previous findings [25]and findings by Sa et al. [26] who also observed similarities regarding Ki-67 expressionbetween humans and dogs.Int. J. Mol. Sci. 2025, 26, 3144 5 of 26Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 5 of 29 Figure 1. Low proliferation and high MECP2 and XPC expression are most pronounced in humans and absent in mice. (A–D). Fluorescent images of oral squamous epithelial from four different species showing MECP2, TP63, XPC (basal cell markers), and MCM2 (proliferation) expression. (E–H). The comparison between basal and suprabasal cells shows that in mice there is no difference in MECP2, XPC and Histone H1 expression, but mouse suprabasal cells are non-proliferative. In humans and dogs, MCM2 expression is mainly found in suprabasal cells. Data shown are from manual multiplex stainings and manual cell segmentation in Leica LASX. Data were exported to Microsoft Excel Version 16.95.1 and Graphpad. * indicates significant difference of p < 0.5 while ns indicates no statistical difference. White bar in immunofluorescence image represents 50 µm. 2.3. Single-Cell Analysis of Basal Cells In Situ: Heterogeneity Within the Basal Cell Layer To expand on our previous description of squamous epithelia in multiple mammalian species by using highly multiplexed immunofluorescence stainings and single-cell analysis, we first established that single-cell analysis allows us to define the basal cell population in situ. In contrast to other approaches such as single-cell RNAseq or flow cytometry, such an in situ multiplex approach retains spatial information as well as cellular and tissue context [27,28]. Comparing the expression of traditional basal cell markers such as ITGB1, KRT15, and EGFR, we evaluated how well the novel basal cell marker XPC depicts basal cells, as indicated in Figure 2A. Using an antibody against KRT10/13 (absent in basal cells), ITGB1 and KRT15 (established basal cell markers) and EGFR (elevated in basal cells), we were able to define the XPC population at the individual cell level (Figure 2A). Identification of individual cells and plotting the expression values of the markers for each cell show that XPC+ cells are ITGB1+ (Figure 2B), KRT15+ (Figure Figure 1. Low proliferation and high MECP2 and XPC expression are most pronounced in humansand absent in mice. (A–D). Fluorescent images of oral squamous epithelial from four different speciesshowing MECP2, TP63, XPC (basal cell markers), and MCM2 (proliferation) expression. (E–H). Thecomparison between basal and suprabasal cells shows that in mice there is no difference in MECP2, XPCand Histone H1 expression, but mouse suprabas l cells are non-proliferative. In humans and d gs, MCM2expression is m inly found in supr b s l cells. Data shown are fr m manual multiplex stainings andmanual cell segmentation in Leica LASX. Data were exported to Microsoft Excel Version 16.95.1 andGraphpad. * indicates significant difference of p < 0.5 while ns indicates no statistical difference. White barin immunofluorescence image represents 50 µm.2.3. Single-Cell Analysis of Basal Cells In Situ: Heterogeneity Within the Basal Cell LayerTo expand on our previous description of squamous epithelia in ultiple mammalianspecies by using highly multiplexed immunofluorescence stainings and single-cell analysis,we first established that single-cell analysis allows us to define the basal cell populationin situ. In contrast to other approaches such as single-cell RNAseq or flow cytometry,such an in situ multiplex approach retains spatial information as well as cellular andtissue context [27,28]. Comparing the expression of traditional basal cell markers suchas ITGB1, KRT15, and EGFR, we evaluated how well the novel basal cell marker XPCdepicts basal cells, as indicated in Figure 2A. Using an antibody against KRT10/13 (absentin basal ells), ITGB1 and KRT15 (established b sal cell markers) and EGFR (elevatedin basal cells), we were able to define the XPC population at the individu l cell level(Figure 2A). Identification of individual cells and plotting the expression values of theInt. J. Mol. Sci. 2025, 26, 3144 6 of 26markers for each cell show that XPC+ cells are ITGB1+ (Figure 2B), KRT15+ (Figure 2C)and KRT10/13neg. (Figure 2D). Using a manual definition of basal cells based on locationand marker expression (highlighted in orange in Figure 2B–G), XPC+ cells were betterable to define “basalness” than KRT15, ITGB1, or EGFR. Of the 85 manually defined basalcells in Figure 2, XPC+ identified 88.24%, ITGB1+ 67.06%, KRT15+ 63.53%, and EGFRhigh62.35% of basal cells; 100% of the cells were double-positive for XPC, and the other basalcell markers were basal cells. In summary, the multiplex approach allows us to better definecell populations in situ. Using XPC as an example of a previously unknown basal cellmarker, we show that substantial heterogeneity within the basal cell population, regardingthe expression of XPC and other basal markers, exists. For example, whether XPClow basalcells represent a special basal subpopulation that is proliferative or is leaving the basal cellpopulation is unclear.Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 6 of 29 2C) and KRT10/13neg. (Figure 2D). Using a manual definition of basal cells based on location and marker expression (highlighted in orange in Figure 2B–G), XPC+ cells w re b tter able to d fine “basalness” than KRT15, ITGB1, or EGFR. Of the 85 manually defined basal cells in Figure 2, XPC+ identified 88.24%, ITGB1+ 67.06%, KRT15+ 63.53%, and EGFRhigh 62.35% of basal cells; 100% of the cells were double-positive for XPC, and the other basal cell markers were basal cells. In summary, the multiplex approach allows us to better define cell populations in situ. Using XPC as an example of a previously unknown basal cell marker, we show that substantial heterogeneity within the basal cell population, regarding the expression of XPC and other basal markers, exists. For example, whether XPClow basal cells represent a special basal subpopulation that is proliferative or is leaving the basal cell population is unclear. Figure 2. Human basal cell heterogeneity based on characteristic markers. (A). Multiplex immunofluorescence staining of human esophageal epithelium exhibiting a distinctly basal cell expression of XPC. (B). A high percentage of basal cells (orange dots) are XPC and ITGB1 positive. (C). Although there is significant overlap between XPC and KRT15 expression in basal cells, subpopulations appear to be XPC, KRT15, or ITGB1 positive. (D). No XPC+ basal cell expresses differentiation marker KRT10 or 13. (E,F). Neither KRT15 nor ITGB1 are enriching basal cell populations as well as XPC; and both can show co-expression with KRT10/13. (G). EGFR is not a specific basal cell marker. Data are derived from manual multiplex stainings and manual cell segmentation (see Supplementary Table S3 for staining outline). Data were exported from Leica Figure 2. Hu an basal cell heterogeneity based on characteristic markers. (A). Multiplex immunoflu-orescence staini g of human esophageal epithelium exhibiting a distinctly basal cell expression ofXPC. (B). A high percentage of bas l cells (orange d ts) are XPC and ITGB1 positive. (C). Althoughthere is significant overlap between XPC and KRT15 expression in basal cells, su populations appearto be XPC, KRT15, or ITGB1 positive. (D). No XPC+ basal cell expresses differentiation marker KRT10or 13. (E,F). Neither KRT15 n ITGB1 are enriching basal cell populatio s as well s XPC; and bothcan show co-expression with KRT10/13. (G). EGFR is n t a specific basal cell marker. Data re derivedfrom manu multiplex stainings an manual cell segmentation (s e Supplementary Table S3 for stainingoutline). Data were exported from L ica LASX a d analyzed in Excel and Graphpad. White bar inimmunofluorescence image represents 50 µm.Int. J. Mol. Sci. 2025, 26, 3144 7 of 262.4. Behavioral Heterogeneity in the Basal Cell Layer: Relationship Between Delaminationand ProliferationAlthough proliferation is low in the human basal cell layer, the parabasal andsuprabasal cells must be replenished to maintain epithelial homeostasis. How this replen-ishment occurs is unclear (Figure 3A). Traditionally, supply of new cells to the suprabasalcell compartment has been discussed in terms of oriented cell divisions using terms suchas planar, perpendicular, and asymmetric cell divisions with a focus on whether a basalcell produces a new basal cell or a suprabasal cell [29,30]. In contrast, delamination hasbeen speculated to be of greater importance in adult squamous epithelia, such as mouseear epidermis. However, in these experiments skin was exposed to a powerful depilatorycream that may have altered cellular behavior [31]. To obtain an overview of the basalcell replenishment in human squamous epithelia versus other species, we counted thenumber of delaminating cells and whether they were proliferating (Ki67 or MCM2 orPCNA positive). We defined delamination histologically (Figure 3B,C). In humans, only afew delaminating cells were proliferating based on Ki67 staining (Table 1); however, thenumbers of MCM2+ and PCNA+ cells, both proliferation markers, indicates that 40% ofthe delaminating cells are potentially proliferative. On the other hand, there is a steepdrop in the basal cell marker MECP2 in delaminating cells to levels seen in parabasal cells(Figure 3(B1)). In dogs, we observed a similar MECP2 expression drop in delaminatingcells (Figure 3(C1)). However, in dogs -in contrast to humans- a similar phenomenon tothe MECP2 expression drop can be observed for p63, whereas in humans p63 expressionis maintained in delaminating cells (Figure 3(B2,C2)). Although most species showed ahigher proliferation rate of delaminating cells than humans, a clear overall abundance ofthese cells was still found mainly in humans, dogs, and mice (Tables 1 and S4). For dogsand mice and to some extent for humans, the findings resemble those in human epidermisby Wang et al. [32] who observed “transitional” basal cells that appear to be in the processof delaminating from the basal layer while proliferating. This suggests that in squamousepithelia, delaminating cells can start to proliferate while being in transition. However,in humans, most delaminating cells did not seem to be actively proliferating based onKi67 staining, a marker that more narrowly identifies proliferating cells in comparisonto PCNA. In summary, delamination is a common mechanism to supply new cells to thesuprabasal cell compartment in many mammalian epithelia and may contribute to thebasal cell heterogeneity we have observed (Figure 2). However, in humans, delaminationmay be more common than in other species and delamination is less frequently associatedwith proliferation.Table 1. Proliferation state of delaminating cells: Using proliferation markers MCM2, PCNA, phospho-histone H3 (PHH3).Species MCM2 % PCNA % PHH3 % Ki67 %Human 40 40 0 20Dog 100 N/A N/A 100Monkey 50 N/A N/A N/AMouse N/A 100 0 40Rat 100 100 0 50Int. J. Mol. Sci. 2025, 26, 3144 8 of 26Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 8 of 29 Figure 3. Quantification and characterization of delaminating cell populations in comparison to adjacent cells. (A). Schematic diagram of mechanisms to deliver cells to the suprabasal cell compartment. (B,C). Example of delaminating cells in human and dog epithelia, respectively. Inserts show exemplary delaminating cells at higher magnification. (B1/C1). Using the basal cell marker MECP2, delaminating cells (D) exhibit a pronounced drop in MECP2 expression compared to adjacent basal cells (B) reaching levels similar to adjacent parabasal cells (P). This observation is true for both humans and dogs. (B2/C2). In contrast to MECP2, p63 expression is not different in B, D and P cells in humans. However, in dogs, p63 expression drops like MECP2. (B3/C3). The intensity of MCM2 expression per B, D, and P cell does not change. White bar in (A) represents 50µm. 2.5. Organization of the Basal Cell Layer in Different Species: Canine Oral Basal Cells Exhibit Two Different Types of Basal Cells To clarify the differences between species in their basal cell characteristics, we used multiplex stainings and defined the relationship between individual markers focusing on human, monkey, dog, and mouse. In humans, there is a clear absence of proliferation in basal cells (represented by orange dots in the scatter plots in Figure 4 MCM2), however this separation of basalness and proliferation is reversed in mice. In monkeys, the basal cells show proliferation (based on marker MCM2) and expression of XPC can be associated with proliferation. In humans, expression of XPC is almost completely excluded from MCM2 positive cells. Therefore, mice and humans represent extremes of squamous epithelial organization. However, in the dog, as alluded to in Figures 1 and 3, the features of basal cells are more complex than in the other analyzed species. To Figure 3. Quantification and characterization of delaminating cell populations in comparison toadjacent cells. (A). Schematic diagram of mechanisms to deliver cells to the suprabasal cell com-partm t. (B,C) Example of delamin ting cells in hum and dog epithelia, respectively. Inse tsshow exemplary delaminating cells at higher magnification. (B1/C1). Using the basal cell markerMECP2, delaminating cells (D) exhibit a pronounced drop in MECP2 expression compared to adjacentbasal cells (B) reaching levels similar to adjacent parabasal cells (P). This observation is true for bothhumans and dogs. (B2/C2). In contrast to MECP2, p63 expression is not different in B, D and P cellsin humans. However, in dogs, p63 expression drops like MECP2. (B3/C3). The intensity of MCM2expression per B, D, d P cell does not change. Whi e bar in (A) represents 50µm.2.5. Organization of the Basal Cell Layer in Different Species: Canine Oral Basal Cells Exhibit TwoDifferent Types of Basal CellsTo clarify the differences between species in their basal cell characteristics, we usedmultiplex stainings and defined the relationship between individual markers focusing onhuman, monkey, dog, and mouse. In humans, there is a clear absence of proliferation inbasal cells (represented by orange dots in the scatter plots in Figure 4 MCM2), however thisseparation of basalness and proliferation is reversed in mice. In monkeys, the basal cellss ow proliferation (based o marker MCM2) and expression of XPC can e associated withproliferation. In humans, expression of XPC is almost completely excluded from MCM2positive cells. Therefore, mice humans represent extremes of squamous epithelialInt. J. Mol. Sci. 2025, 26, 3144 9 of 26organization. However, in the dog, as alluded to in Figures 1 and 3, the features of basalcells are more complex than in the other analyzed species. To untangle this complexity,we performed a 16-plex manual multiplex stain (Supplementary Table S5 for antibodiesand procedure) on normal dog oral mucosa (Figure 5A). Using the Miltenyi MACSiqView1.3 software, we were able to group epithelial cells into 6 clusters using the UMAP tool(Figure 5B,C). Basal cells formed two clusters. One cluster (green cluster in Figure 5C) ischaracterized by high expression in the human quiescent basal cell marker FTH1, whichis related to ferroptosis prevention. The anti-FTH1 antibody works well in both humanand dog tissues. The other cluster is largely FTH1 negative (red cluster in Figure 5C,D).A closer inspection of both clusters and basal cell populations in dogs suggests that thisFTH1-high “Basal 1” population corresponds to cells that resemble human quiescent basalcell layer stem cells (XPC+/ANXA1−/MCM2low) as shown in Figure 5E.Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 9 of 29 untangle this complexity, we performed a 16-plex manual multiplex stain (Supplementary Table S5 for antibodies and procedure) on normal dog oral mucosa (Figure 5A). Using the Miltenyi MACSiqView 1.3 software, we were able to group pithelial cells into 6 clusters using the UMAP tool (Figure 5B,C). Basal cells formed two clusters. One cluster (green cluster in Figure 5C) is characterized by high expression in the human quiescent basal cell marker FTH1, which is elated to ferroptosis prevention. The anti-FTH1 antibody works well in both human and dog tissues. The other cluster is largely FTH1 negative (red cluster in Figure 5C,D). A closer inspection of both clusters and basal cell populations in dogs suggests that this FTH1-high “Basal 1” population corresponds to cells that resemble human quiescent basal cell layer stem cells (XPC+/ANXA1-/MCM2low) as shown in Figure 5E. Figure 4. Characterization of basal cells versus suprabasal cells of multiple species using basal cell markers XPC, MECP2, p63 and proliferation marker MCM2. Manual multiplex stainings were analyzed by manually identifying cells and measuring their expression levels using Leica LASX version 3.7 software. Figure 4. Characterization of basal cells versus suprabasal cells of multiple species using basalcell markers XPC, MECP2, p63 and proliferation marker MCM2. Manual multiplex stainings wereanalyzed by manually identifying cells and measuring their expression levels using Leica LASXversion 3.7 software.Int. J. Mol. Sci. 2025, 26, 3144 10 of 26Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 10 of 29 Figure 5. Analysis of canine basal cell populations and their similarities to the human condition. (A). Example of dog oral mucosa 16-plex multiplex stain showing 5 markers. (B,C). UMAP clustering of epithelial cells shows 6 cell populations including 2 basal cell populations (green and red). (D). Heatmap of FTH1 expression within UMAP shows enhanced expression in “green” basal cell population. (E). Definition of “green” basal cells (Basal 1) as cells similar to human basal cells. Data are derived from manual multiplex stainings (see Supplementary Table S5). Confocal images were exported and used in MACSiQView to cluster cell populations using UMAP. To generate box plots, data were exported from MACSiQView and analyzed in Graphpad. * indicates significant difference of p < 0.5 while ns indicates no statistical difference. White bar in (A) indicates 50 µm. 2.6. Comparison of scRNAseq Datasets Confirms Human and Mouse Differences in Basal Cell Proliferation and Proliferation-Associated TGFbeta, WNT, and SHH Pathways To define whether proliferation is the main difference between mouse and human basal cell layer cells, we next compared existing single-cell RNAseq (scRNA-seq) data from mouse and human squamous epithelia. Although the studies included in our comparison vary greatly in their basal cell marker sets and show limited overlap (Figure 6A; see Supplementary Table S6), key characteristics that consistently distinguish human from mouse basal cells are enhanced “TGF-beta regulation of extracellular matrix” “Cytokine Signaling” and “NRF2 Pathway”/”Ferroptosis” signatures. For examples of ferroptosis-related protein expression in the human basal cell layer, see Supplementary Figure S3 and Supplementary Table S7. As expected, the main characteristic of the 245 Figure 5. Analysis of canine basal cell populations and their similarities to the human condition. (A).Example of dog oral mucosa 16-plex multiplex stain showing 5 markers. (B,C). UMAP clusteringof epithelial cells sh ws 6 cell populations including 2 basal cell populations (green and red). (D).Heatmap of FTH1 expression within UMAP shows enhanced expression in “green” basal cell pop-ulation. (E). Definition of “green” basal cells (Basal 1) as cells similar to human basal cells. Dataare derived from manual multiplex stainings (see Supplementary Table S5). Confocal images wereexported and used in MACSiQView to cluster cell populations using UMAP. To generate box plots,data were exported from MACSiQView and analyzed in Graphpad. * indicates significant differenceof p < 0.5 while ns indicates no statistical difference. White bar in (A) indicates 50 µm.Int. J. Mol. Sci. 2025, 26, 3144 11 of 262.6. Comparison of scRNAseq Datasets Confirms Human and Mouse Differences in Basal CellProliferation and Proliferation-Associated TGFbeta, WNT, and SHH PathwaysTo define whether proliferation is the main difference between mouse and humanbasal cell layer cells, we next compared existing single-cell RNAseq (scRNA-seq) data frommouse and human squamous epithelia. Although the studies included in our comparisonvary greatly in their basal cell marker sets and show limited overlap (Figure 6A; seeSupplementary Table S6), key characteristics that consistently distinguish human frommouse basal cells are enhanced “TGF-beta regulation of extracellular matrix” “CytokineSignaling” and “NRF2 Pathway”/”Ferroptosis” signatures. For examples of ferroptosis-related protein expression in the human basal cell layer, see Supplementary Figure S3 andSupplementary Table S7. As expected, the main characteristic of the 245 mouse-specificbasal cell layer markers (Figure 6B) that are absent in humans, is their involvement inregulation of cell proliferation (e.g., KEGG term “Cell Cycle”; Supplementary Table S6).Beyond proliferation, the mouse data indicate enhanced Hedgehog and WNT SignalingPathway signatures, as well as a small “Cysteine and methionine metabolism” relatedgene signature (Figure 6B, mouse “specific” genes include cyclins and other proliferationmarkers; Supplementary Table S6).Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 11 of 29 mouse-specific basal cell layer markers (Figure 6B) that are absent in humans, is their involvement in regulation of cell proliferation (e.g., KEGG term “Cell Cycle”; Supplementary Table S6). Beyond proliferation, the mouse data indicate enhanced Hedgehog and WNT Signaling Pathway signatures, as well as a small “Cysteine and methionine metabolism” related gene signature (Figure 6B, mouse “specific” genes include cyclins and other proliferation markers; Supplementary Table S6). Figure 6. Determining human specific basal cell gene expression. (A). Venn diagram to identify human basal cell markers based on scRNAseq datasets. (B). Commonalities and differences in mouse and human basal cell markers: highlighting a role of NRF2 signaling, ferroptosis, and a lack of DNA repair related basal markers XPC and MECP2 in scRNAseq data. (C). In humans, basal cells (white arrowheads) express little EZH2. EZH2 (red) is mainly expressed in proliferating (PCNA+ green) human parabasal cells. (D). In contrast, in mice EZH2 (red) is expressed in proliferating (PCNA, green) basal cells (white arrows). (E,F). CAV1 is expressed in basal cells (white arrows) in both humans and mice. (G,H). ASS1 is expressed in basal cells (white arrows) in both humans and mice. White bar in (H) represents 50 µm and can be applied to (C–F) as well. In addition, considering target genes of transcription factors from published ChIP datasets (ChEA database) human basal cells tend to have an overrepresentation of the anti-proliferative SMAD2/3/4 target genes [25] while mouse basal cells show an overrepresentation of proliferation associated FOXM1 target genes (Supplementary Table S6). Overall, these data confirm our previous findings ([5,25], Figures 1 and 4) and corroborate a different organization of the mouse basal cell layer compared to the human basal cell layer. Figure 6. Determining human specific basal cell gene expression. (A). Venn diagram to identifyhuman basal cell markers based on scRNAseq datasets. (B). Commonalities and differences in mouseand hum n basal cell markers: highlighting a role of NRF2 signaling, ferroptosis, and a lack of DNArepair related basal markers XPC and MECP2 in scRNAseq data. (C). In humans, basal cells (whitearrowheads) express little EZH2. EZH2 (re ) is mainly express d in proliferating (PCNA+ green)human parabasal cells. (D). In contrast, in mice EZH2 (red) is expressed in proliferating (P ,green) basal cells (white arrows). (E,F). CAV1 is expressed in basal cells (white arrows) in bothhumans and mice. (G,H). ASS1 is expressed in basal cells (white arrows) in both humans and mice.White bar in (H) represents 50 µm and can be applied to (C–F) as well.Int. J. Mol. Sci. 2025, 26, 3144 12 of 26In addition, considering target genes of transcription factors from published ChIPdatasets (ChEA database) human basal cells tend to have an overrepresentation of theanti-proliferative SMAD2/3/4 target genes [25] while mouse basal cells show an over-representation of proliferation associated FOXM1 target genes (Supplementary Table S6).Overall, these data confirm our previous findings ([5,25], Figures 1 and 4) and corroborate adifferent organization of the mouse basal cell layer compared to the human basal cell layer.2.7. XPC and MECP2 Interaction Is Restricted to Quiescent Basal CellsA human enhanced “pathway” that does remain undetected in the scRNAseqcomparison, likely due to either low mRNA expression or posttranscriptional regula-tion, is genome protection via elevated DNA repair. Two proteins, XPC and MECP2,stand out because of their role in DNA repair [20,33–37], their basal cell co-expression(see Figures 1 and 4), and their direct interaction with each other [19]. Given that thepattern and intensity of MECP2/XPC co-expression differ between mice and humans(Figures 1–4), we questioned whether this co-expression is a characteristic feature of humanstem cells, contributing to their health and potentially playing a tumor-suppressive roleby preventing squamous cell carcinoma and its precursors. We speculate that MECP2 andXPC expression is lost during tumorigenesis because of their putative keratinocyte stemcell–protective functions [38]. To test this hypothesis, we set out to determine: 1. Whetherthe expression and interaction of MECP2 and XPC are altered in precancerous lesions, and2. at what stage the quiescent basal cell layer—putatively tumor-suppressive—is disruptedduring the normal-precancer-cancer progression, and how this disruption affects basalmarker expression.If MECP2/XPC expression and basal cell quiescence are indeed tumor-suppressive(and linked to longevity), then we would expect a coordinated loss of MECP2, XPC, andbasal cell quiescence during the transition to precancer. Since a key factor for stem cellquiescence is the reduction in replication errors, any impairment in this process couldcontribute to tumorigenesis.To explore these ideas, we performed a combination of prox-imity ligation assays (PLA) and MECP2/XPC immunofluorescence stainings to showinteraction between MECP2 and XPC, specifically in normal human quiescent basal cellswhich become progressively lost during progression from normal to cancer (Figure 7A,Supplementary Table S8 for overview of procedure). In normal human squamous epithelia(in this case of the esophagus), PLA signal is restricted to basal cells (Figure 7A, quantifi-cation 7B). However, while XPC and MECP2 have strong signals in the basal cell layer,based on the PLA signal, the interaction between these two proteins appears limited (seeSupplementary Figure S4 for additional information). To explore whether the interaction isan indicator for repair, we hypothesized that the PLA signal is correlated with proliferationrate, extent of inflammation, or p53 expression. Such signs of epithelial activation andstress may be accompanied by increased DNA damage (e.g., by oxidative stress). However,we could not detect any correlation with such features and PLA signal intensity. Althoughwe could detect a reduction in PLA, MECP2 and XPC signal (Figure 7B–D) during progres-sion from normal to cancer, we also observed a slight “recovery” of the PLA signal in thecancer samples (SCC in Figure 7B compared to high-grade dysplasias [HGD] lesions). Thissuggests a change in MECP2–XPC dynamics or a change in the frequency of the underlyingcause for MECP2–XPC interactions.Int. J. Mol. Sci. 2025, 26, 3144 13 of 26Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 13 of 29 Figure 7. Proximity ligation assay (PLA)-based analysis of MECP2–XPC interactions. (A). Representative PLA examples depicting interactions specifically in human basal cells but loss in human esophageal dysplastic lesions and SCC. PLA signal occurs as orange dots and some of them are highlighted with orange arrows on epithelial cells. White arrows indicate stromal artifacts. To define basal cells several markers were stained subsequent to the PLA procedure including SLC3A2, KRT10/13, and Ki67 (see Supplementary Figure S4, which also contains information about control PLA experiments). Below the PLA image are hematoxylin and eosin (H&E) stains from the same area. Black bar in the H&E stain represents 50 µm. (B). Quantification of PLAs showing a significant reduction in PLA signal from normal basal cells (N) to suprabasal cells (NS) in normal squamous epithelial. A similar or even more pronounced drop in PLA signal is observed during the progression from normal (N) to squamous cell carcinoma (SCC). Student’s t test shows a statistical reduction in PLA signal between NB and the following states: NS, LGD (low grade dysplasia), HGD (high grade dysplasia), but not SCC. (C,D). MECP2 and XPC expression, respectively, are reduced similar to the PLA signal from normal to dysplasia and SCC. Student’s t test shows a statistical reduction in MECP2 as well as XPC expression between NB and the following states: HGD and SCC. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. N, normal basal cells from Figure 7. Proximity ligation assay (PLA)-based analysis of MECP2–XPC interactions. (A). Repre-sentative PLA examples depicting interactions specifically in human basal cells but loss in humanesophageal dysplastic lesions and SCC. PLA signal occurs as orange dots and some of them arehighlighted with orange arrows on epithelial cells. White arrows indicate stromal artifacts. Todefine basal cells several markers were stained subsequent to the PLA procedure including SLC3A2,KRT10/13, and Ki67 (see Supplementary Figure S4, which also contains information about controlPLA experiments). Below the PLA image are hematoxylin and eosin (H&E) stains from the samearea. Black bar in the H&E stain represents 50 µm. (B). Quantification of PLAs showing a significantreduction in PLA signal from normal basal cells (N) to suprabasal cells (NS) in normal squamousepithelial. A similar or even more pronounced drop in PLA signal is observed during the progressionfrom normal (N) to squamous cell carcinoma (SCC). Student’s t test shows a statistical reduction inPLA signal between NB and the following states: NS, LGD (low grade dysplasia), HGD (high gradedysplasia), but not SCC. (C,D). MECP2 and XPC expression, respectively, are reduced similar to thePLA signal from normal to dysplasia and SCC. Student’s t test shows a statistical reduction in MECP2as well as XPC expression between NB and the following states: HGD and SCC. Statistical signifi-cance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. N, normal basal cells from 25 samples; NS,normal suprabasal cells from 25 samples; LGD, low-grade dysplasia basal cells from 14 lesions; HGD,basal like cells from 19 high-grade dyspalsias; SCC, basal like cells from 9 SCC samples. Analysissolely based on cells rather than averages for each sample are provided in Supplementary Figure S4.All samples from TMA BBS02011, imaged on a Leica SP5 microscope, analyzed in MACSiqView, andgraphs generated in Prism.Int. J. Mol. Sci. 2025, 26, 3144 14 of 26In summary, the levels of XPC, MECP2, and XPC–MECP2 interactions decrease mostdrastically at the LGD-HGD transition.3. DiscussionComparing mammalian species of different maximum lifespans suggests widespreadgene expression changes associated with pathways guarding tissue integrity and ensurescellular protection. Here, we have focused on one specific tissue type that is generally notrepresented in comparative analyses of mammalian biology [39–44], i.e., self-renewingsquamous epithelia. Such epithelia are of special interest to the biology of longevity due totheir relatively high proliferation and turn-over rate, their exposure to external stress factors,and their elevated age- and carcinogen-dependent risk for malignant transformation (e.g.,most human cancers are carcinomas).The mouse has served as the primary genetic model for studying squamous epithelia.However, it has become clear that the mouse shows “marked differences in the cellularheterogeneity” compared to humans [45]. Key differences are the quiescence of the humanbasal cell layer stem cells and the shift in proliferation into the parabasal cell population[5,25]. To better understand this peculiar human feature in the context of longevity, wehave used an in situ proteomic approach.3.1. The Meaning of Stem Cell QuiescenceOur theory that the observed slow-cycling/quiescent human basal cell layer is anadaptation to human longevity is supported by comparative studies of intestinal stemcells in naked mole rats [46]. In their words, “adaptations of adult stem cells. . .have oneof the greatest impacts on organismal health and lifespan.” According to Montazid et al.,adaptation to longevity in naked mole rats involves several modifications compared to mice:expansion of the size of the stem cell compartment, a slowdown in stem cell kinetics, anda turnover rate that more closely resembles humans rather than mice. Consequently, thereduced cell division rate of naked mole rat stem cells results in reduced somatic mutationrates [44]. In addition to the reduced cell divisions within the stem cell compartment, nakedmole rats also show increased resistance to stress [47], a feature closely associated withlongevity [48]. Therefore, the combination we have observed in human squamous epitheliastem cells of a reduced cell division rate and the increased expression of cytoprotectiveproteins that mediate stress-resistance [5] may form the backbone of adaptation to longevityand provides a flexible tool for evolution to modify cell behavior. This is in line with ourobservations of a spectrum of parabasal proliferation ranging from absent in mice to ahybrid form in monkeys, dolphins and dogs, and reaching its purest form in humans [25].Another example of a species-specific difference in stem cell organization found inspermatogonial stem cell populations, where adult human (but not rodent) cells main-tain a fetal-like signature—a feature the authors interpret as an adaptation to humanlongevity [49].3.2. Alternative ExplanationsVarious explanations for the variable stem cell quiescence observed in different speciesthat do not involve adaptation to longevity can be discussed; however, these alternativeslack the conclusiveness of the longevity adaptation hypothesis. The vast majority of theliterature interprets stem cell quiescence as a protective mechanism to prevent cancer andtissue dysfunction [50,51]. Nonetheless, a quiescent stem cell layer may possess otherfeatures that could have driven its evolution, such as mechanical stress adaptation to helpthe oral epithelium withstand chewing certain foods. However, the general solution tosuch mechanical stress is typically keratinization and the formation of rete ridges, whichInt. J. Mol. Sci. 2025, 26, 3144 15 of 26are well-documented structural adaptations in various epithelial tissues, including oralepithelium, across mammals [52,53].Alternatively, a quiescent stem cell layer could facilitate specialized niche signalingthat is absent in most other species. However, there is no clear evidence that the nicheenvironment of human squamous epithelium is fundamentally different from that inother mammals, nor that quiescent cells possess intrinsically different signaling functionscompared to proliferating cells [54].Human quiescent basal cells may also engage in specific immune system modulation,minimizing immune activation and chronic inflammation through specialized interactionswith immune cells. Even if such an immune-modulatory function were unique to humanquiescent basal cells, the resulting reduction in inflammation would inherently contributeto the longevity of the epithelium and, consequently, the organism. However, evidencesupporting unique immune interactions specific to human quiescent stem cells especialsquamous basal cells remains limited [55,56].Another potential explanation is the protection against viral integration, particularlyrelevant in squamous epithelia of the Waldeyer’s ring area and the cervix. Low proliferationrates may reduce opportunities for viral integration and infection in basal cells, therebydecreasing the risk of developing squamous cell carcinomas. Nevertheless, viruses canintegrate into both quiescent and proliferative cells, and other antiviral defense mecha-nisms exist that do not rely solely on reducing cell proliferation (one remotely relevantexample: [57]).Quiescence is often associated with a specific metabolic state [58]. One could arguethat quiescent basal cells may have specialized metabolic profiles that support their long-term maintenance. However, long-term maintenance is intrinsically linked to longevity,suggesting that this adaptation ultimately supports our theory that the human basal celllayer represents an adaptation to longevity.We have observed a distinct association of Polycomb proteins and other epigeneticregulators with basal and parabasal cells [25]. This could indicate that unique epigeneticlandscapes in quiescent basal cells maintain their identity and functionality. However, epi-genetic regulation is a common mechanism across various stem cell populations, regardlessof their proliferation status, and does not inherently require quiescence. Thus, this too canbe viewed as another facet of the longevity adaptation hypothesis.Finally, one could argue that the observed stem cell quiescence in the basal cell layerenhances differentiation control, ensuring precise differentiation of stem cells and main-taining epithelial integrity and functionality. However, the exact mechanisms underlyingthis enhanced differentiation system remain unclear. Moreover, it is perplexing why such asystem is not employed by other mammals, given their similar requirements for epithelialmaintenance [59].In summary, while alternative hypotheses offer potential explanations for the presenceof a highly quiescent basal cell layer in human squamous epithelium, they either lack em-pirical support or overlap significantly with the primary longevity adaptation hypothesis.Therefore, the simplest and most straightforward explanation for the observed spectrum ofbasal quiescence and parabasal proliferation in mammals is an adaptation to longevity [25].This corresponds well with the observation that lifetime stem cell divisions correlate withcancer risk [3]. Whether squamous epithelial stem cell quiescence is a uniquely humanadaptation to longevity has to be seen. Recently Zhang and Gines stated that “it seemsthat every species studied so far has its unique pathway of cancer resistance because ofdifferent life histories and selective pressures” [60]. This suggest that studying species withvastly different longevity may produce valuable insights into how to protect humans betterfrom cancer.Int. J. Mol. Sci. 2025, 26, 3144 16 of 263.3. Proteomic In Situ Analysis Can Detect Putative Features of Longevity in Species Comparisons:XPC and MECP2It has been speculated that one mechanism to enhance cell, particularly stem cell,cytoprotection is the enhanced expression of DNA repair genes. The elevated expression ofDNA repair genes may contribute to increased longevity. Our analysis has focused on twogenes associated with DNA repair, XPC [33] and MECP2 [34,37,61]. The expression of bothproteins is elevated in human basal cells compared to epithelial cells in other mammals,especially rodents (ca. 5-11-fold up, Figure 1). Furthermore, the elevated expression of bothproteins is associated with a specialized quiescent stem cell layer in humans, a cell layerthat is absent in rodents. It is well documented that loss of XPC reduces life expectancyand causes skin cancers. Furthermore, in vitro XPC KO cells are highly sensitive to DNAdamaging agents due to impaired DNA repair [38].There is accumulating evidence that MECP2 plays a role in controlling oxidative stressand DNA damage [34,36,61–65]. This suggests that due to MECP2′s interaction with XPCand its specific location in epithelial stem cells, it could have cell protective functions.It is interesting to note that the main type of mutation driving somatic mutationrates are C>T mutations due to deamination of 5′methylcytosine [44]. MECP2 is a pro-tein that binds to sites of DNA methylation and CpG islands [66]. It is unclear whetherMECP2 can protect against this specific mutation event, but a close relative, MBD4, hasbeen suggested to function as a “safeguard against damage from 5mC deamination” [67].However, MECP2 lacks the DNA glycosylase domain of MBD4 and therefore it is unclearhow MECP2 may contribute to the protection of methylated CpG sites. Nevertheless,MECP2 has the ability to interact with a “mastermind” of DNA repair, XPC [37] whilesuch interactions are not documented for MBD4. Whether this XPC–MECP2 interaction iscrucial for maintaining stem cell DNA integrity remains to be seen. Interestingly, the lossof both MECP2 and XPC expression coincides with the formation of high-grade dysplastic(HGD) lesions (Figure 7C,D). This step coincides with the loss of the quiescent basal celllayer in precancer [68–71].3.4. XPC-MECP2 Interactions Are Limited to Quiescent Basal CellsOur in situ spatial analysis also allowed us to demonstrate a direct interaction betweenXPC and MECP2 that had been alluded to by previous studies [37]. Using a proximityligation assay (PLA), the XPC–MECP2 interaction occurs specifically in quiescent basal cells.The number of PLA dots is relatively low in these basal cells, suggesting that only a smallportion of MECP2 and XPC molecules interact. This interaction may represent a repairevent which involves both MECP2 and XPC [37]. Since DNA damage occurs thousandsof times per day per cell [72–74], MECP2 and XPC may provide a specific surveillanceand repair system that is low in the non-stem cells of human squamous epithelia and isalso low in rodent squamous epithelial cells. One of the functions of XPC is in NucleotideExcision Repair (NER) in response to distortions in the DNA helix due to “bulky” DNAdamage. Although it is difficult to estimate exact numbers, such DNA damage eventsinvolving XPC and NER may occur in the range of 10–102 per day per cell, which couldreflect the numbers of PLA dots we observed on 5 µm thick sections. Furthermore, XPCalso participates in “smaller” DNA damage repair and can contribute to Base ExcisionRepair (BER; [75]). XPC may function as a “general sensor of damaged DNA” [76]. BERis likely to happen frequently in human cells and may not fit the low numbers of PLAsignals per basal cell. On the other hand, basal cells have an elevated anti-oxidative damageprogram that could strongly reduce events requiring BER.Alternatively, we observed PLA signals could represent random MECP2 and XPCinteractions. If this were the case, we should expect a strict correlation between PLA signalInt. J. Mol. Sci. 2025, 26, 3144 17 of 26and MECP2 and XPC expression. Although that is generally true, e.g., there is a drop inPLA signal from basal to suprabasal cells coinciding with a strong reduction in MECP2and XPC expression (Figure 7). However, in SCC cells we observed a slight recovery of thePLA signal compared to high-grade dysplastic lesions (HGD, Figure 7B). This suggests thateven in SCC cells with lower MECP2 and XPC expression, interactions between these twoproteins still manifest. Our hypothesis is that this increase in interaction is specifically dueto increased DNA damage burden in SCC cells. In summary, we observe a reduction in PLAsignal associated with progression towards cancer. Whether this loss is associated withthe loss of quiescent stem cell features or whether it is just due to the loss of MECP2 andXPC expression levels remains to be shown. Further investigations are required to dissectthe significance of MECP2 and XPC interactions and how they change during progressiontowards cancer.3.5. Enriched Stem Cell Pathways in Human Squamous EpitheliaTo address what may make the human basal cell layer special beyond low proliferationrates, we performed comparisons between existing scRNAseq datasets from mice andhumans. As summarized in Table 2, the markers present in at least two human scRNAseqdatasets and in our proteomic dataset, can be interpreted as indicators of readiness, such asmetabolic changes, infection, and wounding. Consistent with basal cells being attached tothe basal lamina, one subset of markers is associated with this “household” function.Table 2. Selected high-quality basal cell markers and their putative stem cell functions.Basal Cell Marker Function Putative Role in Basal Cells Related Basal ProteinsABCC3 cellular waste export of often GSHconjugated molecules; detoxificationenhanced protection againstcytotoxic substances. GSR, GSSASS1 urea cycle; arginine synthesisWithout other enzymes of the urea cycle, ASS1cannot make arginine. However, NOS1 couldmake arginine into citrulline and ASS1 coulduse this as substrate to start makingarginine again.SLC3A2CAV1scaffolding protein of caveolae plasmamembranes; regulatory function ofendocytosis, antiproliferativeenhance adhesion to basal lamina; maysequester growth factor receptors and deepenquiescence; in caveolae: absorbing mechanicalstress and preventing cell damage (membranereservoir); may contribute to deep quiescencedue to its antiproliferative effects;differentiation inhibitor;may have link to ferroptosis.CAV2, CAVIN1, FLOT1, BSG,DST, ITGB1, ITGA6, LIMA1,NGFR, SLC1A3, SLC3A2,SLC7A5, CDH13CAVIN1 scaffolding protein of caveolaeplasma membranesenhance adhesion to basal lamina; maysequester growth factor receptors and deepenquiescence; in caveolae: absorbing mechanicalstress and preventing cell damage.CAV1, CAV2, FLOT1CDH13cell membrane and is anchored by aGPI moiety, rather than by atransmembrane domainmay function as an adiponectin receptor?Tumor suppressor? ITGB1COL17A1 attachment of basal keratinocytes tothe underlying basement membrane“household” protein to adherebasal cells to the basal lamina. DST, LAMC1, ITGB1, ITGB4,CXCL14 chemoattractant for immune cells;may mediate stromal reprogrammingmay mediate antimicrobial activity; problem:how is it kept in an inactive state beforeneeded? Main target may be fibroblasts whichexpress high levels of the putative CXCL14receptors LRP1 or GPR85. May also berequired for the recruitment of dendritic cellsand macrophages to sites of infection orinflammation but CXCL4 ko mice have nophenotype; potential anti-tumor activity.Enigma.CYBRD1 Iron metabolism, iron uptakeFerroptosis? Iron metabolism related toFTH1/FTL: reduces iron forstorage by ferritin?FLOT1, LIMA1, FTH1Int. J. Mol. Sci. 2025, 26, 3144 18 of 26Table 2. Cont.Basal Cell Marker Function Putative Role in Basal Cells Related Basal ProteinsDKK3only receptor for DKK3 is CKAP4. CKAP4mainly expressed on differentiatedkeratinocytes, fibroblasts.DST hemidesmosome linker tocytoskeleton“household” protein to adhere basal cells tothe basal lamina COL17A1, KRT14FAT1 cell–cell adhesion, cell polarity, WNTand Hippo signalingtumor suppressor function may provide asafety feature for stem cells. LATS1, YAP1FBXO2binds denatured glycoproteins,preferentially those of thehigh-mannose typeDefense: detection of bacterial surface sugarsand viral glycoproteins? Maintenance:Clearance of damaged lysosomes?FHL2 scaffolding protein, interaction withdozens of proteins,maybe advantageous to maintain ER health?Enigma.IFITM3 prevents the fusion of viralmembranes with host cell membraneswithout infection: maintain membranestability and integrity, Readiness for viralinfection and turning of interferon response.Elevated level of readiness. Saves time toreach high enough IFITM3 levels to preventviral entry.IFITM1, IFITM2, TLR3, CAV1,IL18 cytokinemay be “stored” as precursor pro-IL18 to makethe cell ready for an emergency and accelerateresponse times; hypothesis: IL18 exists in itspro-form (supported by PMID: 37068092).IFITM1, IFITM2, IFITM3IL1R2 decoy receptor for IL1Dampens inflammatory signaling; maybeprotects basal stem cells from activation by“low” level inflammatory signals andmaintains a quiescent state even ininflammation (see PMID: 38637492).ITGB4 hemidesmosomes “household” protein to adherebasal cells to the basal lamina.ITGA6, LAMB2,LAMB3, LAMC1, LAMC2;COL17A1, DST, KRT14KRT14 Intermediate filament cytoskeleton “household” protein to adherebasal cells to the basal lamina.ITGA6, LAMB2,LAMB3, LAMC1, LAMC2;COL17A1, DST, KRT14KRT19 Intermediate filament cytoskeleton “household” protein to adherebasal cells to the basal lamina.LAMB3 basal lamina “household” protein to adherebasal cells to the basal lamina.MOXD1 unknown Enigma.MXRA5TGF-β1-regulated withanti-inflammatory andanti-fibrotic propertiesNFIB Transcriptional regulatorInhibits EZH2, a marker of parabasal cells.May “govern super-enhancer maintenance”specific for quiescent basal stem cells.SOX2, SOX6NINJI1 anti-bacterial host defense byplasma membrane ruptureMay supercharge inflammation in case ofcatastrophic infection of stem cells.NTRK2 BDNF receptor Enigma. DST, NGFRPCDH7 cell–cell-adhesion, RHO signaling MECP2, AHRGEF28SH3PXD2A scaffolding protein prepares a cell formigration, invasionpotentially provides readiness for cellmigration. FSCN1, LIMA1SLC1A3 high affinity glutamate transporterglutamate can be a substrate to make otheramino acids or made into glutamine forammonia detoxification by GLUL.GLULSLC1A4neutral amino-acid transporter thatmediates transport of alanine, serine,cysteine, proline, hydroxyprolineand threonine.Cysteine uptake for GSH production. GSR, GSSSLC7A5 uptake of large neutral amino acids may import amino acids andactivate mTOR pathway. SLC3A2SOX6 transcription factorgrowth inhibitor; gene targets unknown thatmay mediate quiescence; may regulateextracellular matrix gene expression;unknown partner in quiescent basal cells.BCL7A, FLOT1, NFIB,NFIC, SOX2, ZNF503TNS1 cell–matrix adhesion “household” protein to adhere basal cells tothe basal lamina. ITGB1SUMMARY1. Household function in cell–matrixadhesion. 2. Cell protection.3.Many genes that by themselvesappear “useless”.Explanations: 1. readiness for emergenciesregarding wounding and infection, nutrientdeficiencies; 2. feeder cell function forsuprabasal cells (large set of transportermolecules); 3. enhances cellular protection.IL18, CXCL14, IFITM3, GSR,GSS, SLC3A2, SLC7A5,SLC7A8, SLC1A3, ASS1Int. J. Mol. Sci. 2025, 26, 3144 19 of 26For example, ASS1, a critical enzyme in the urea cycle, is a component of the quiescentbasal cell proteome difficult to comprehend. However, most non-hepatic cells do notexpress most urea cycle enzymes. For example, there is no indication for OTC and ARG1gene expression in quiescent basal cells of squamous epithelia. High ASS1 levels mayprovide basal cells high flexibility in case of arginine deprivation and usage of citrulline.What conditions may require ASS1 activity in squamous basal cells is unknown. Thisexample highlights that several basal cell markers have no clear-cut function unless stressand emergency conditions are considered.3.6. A Combo of Protective Features to Enhance Stem Cell Resilience?Beyond DNA repair related genes (e.g., the aforementioned XPC, MECP2 and RAD23B,PML, DDB2), our analysis suggests the existence of a set of human specific (absent in mouse)basal stem cell markers that also includes NRF2-related anti-ferroptosis genes (ABCC3,CBR1, SLC6A15, GSR, SLC39A13, SLC2A5, PRNP, SLC40A1), and defense genes (e.g.,IFITM3, CD74, CD40; IFITM2, ERAP2, IL18, IFIT3, PML, GHR, BST2).This elevated emphasis on cytoprotection in a human stem cell population may bethe foundation of low mutation rates per bp per year in humans in general. For example,humans have the lowest substitution rates per genome per year in a comparative studyon colon samples [44]. How the somatic mutation rate per year is reduced almost 17-times in humans compared to mice is unclear [44]. We know little about the stem cellorganization in the colon in different species. Whether there is a dedicated quiescent stemcell population in human colonic crypts that is absent in other species, is also unknown. Theauthors concluded that in the colon, the mutations are driven by, “endogenous mutationalprocesses . . . including 5-methylcytosine deamination and oxidative damage” [44] andthat, “cell division rates are not a major determinant of somatic mutation rates acrosshuman tissues.” However, this assumption that the somatic mutation rate is independentof the number of cell divisions is misleading and the data of Abascal et al. [77] actuallyshow a 2-3-fold variation in mutation rates and the highest mutation rate in the cell typewith the highest proliferation rate (i.e., the colon). The idea that “modest” differences inmutation rate due to differences in cell division numbers are irrelevant to the risk of cancer,appears hard to sustain. A two-fold increase in mutation rate may make certain tissues the“weakest link” and cause problems in the long run. For example, the mere 2-3-fold increasein mutation rates increases the cancer risk of patients with MUTYH [78], and 1.2-7-foldfor POLE and POLD1 mutations [79]. Surprisingly, the increased mutation rate in patientswith germline mutations in these genes do not show overt premature aging phenotypes.Furthermore, mutational burden is associated with origins of replication [80,81].In this context, we also would like to highlight that the cytoprotective quiescent state ofthe basal stem cells is extremely stable and only broken in extreme conditions [5]. Recently,studies on inflammatory states have confirmed this robustness of “deep” quiescence inthe human esophagus by Rochman et al. observing that inflammation leads to “hyper-proliferation of less mature epithelial subpopulations yet stability of the quiescent, stemcell–enriched basal epithelial compartment” [82].3.7. Limitations of the StudyWhile we have discussed several explanations for the presence of the human basalquiescent stem cell layer (see Sections 3.1 and 3.2), we cannot rule out that factors unrelatedto longevity adaptation have influenced the development of this anatomical feature. A keylimitation is that our study did not cover all mammalian species. Out of approximately6400 known living mammalian species, we investigated only 16—omitting many speciesnoted for extreme longevity. However, we did include several rodents (including mice),Int. J. Mol. Sci. 2025, 26, 3144 20 of 26and our longevity distribution roughly matches that observed across all mammals (seebottom of Supplementary Table S1).Consequently, determining how “unique” human stem cell organization is remainschallenging. Interestingly, we observed no obvious differences in stem cell proliferation andbasal cell organization between long-lived rodents—such as Spalax ehrenberg and Sciuruscarolinensis (each with a maximum lifespan above 20 years)—and shorter-lived specieslike mice and rats (here and [25]). This suggests that adaptation to longevity is not aone-size-fits-all process.Additionally, our study examined how the progression from normal tissue to dysplasiato cancer impacts basal stem cell layer quiescence, and we explored the point at whichnormal stem cell behavior might be lost during human squamous cell carcinogenesis.However, we do not yet understand whether or how normal stem cells transform intocancer stem cells (CSCs). Our data indicate that a critical step toward the loss of normalstemness and basal cell quiescence likely occurs in high-grade dysplasias (HGDs)—apathological state associated with the disruption of a normal basal cell phenotype [68–71].This change may represent an important step toward the elusive acquisition of invasivebehavior, a molecular event that remains surprisingly poorly understood [83].4. Materials and Methods4.1. Antibody ListSee Supplementary Tables S1, S3, S5 and S8.4.2. Antibody Evaluation for SpeciesTo determine whether an antibody “works” to detect its target epitope in a specificspecies, we used the following criteria: 1. The staining patterns are as expected andreflects human protein expression patterns based on the literature, our experience, andthe Human Protein Atlas, 2. If information on the epitope is available, we compared suchprotein sequence data from different species, 3. Checking antibody supplier data for theirrecommended use for which species, 4. Exploring the publication record for the antibodyand its use in different species. These data are documented in Supplementary Table S1.4.3. Manual Multiplex Cyclic Immunofluorescence (mcyIF) Stainings5 µM formalin-fixed, paraffin-embedded (FFPE) tissue sections were deparaffinizedwith xylenes, rehydrated, and antigen retrieval was achieved by incubating for 13 minin 1xTE in a Cuisinart pressure cooker [84,85] and remaining in the pressure cooker foranother 10 min. After removing from the pressure cooker, slides were cooled down for20 min and incubated simultaneously with two to three primary antibodies that are directlyconjugated with AlexaFluor488 or FITC, AlexaFluor555 or PE, and AlexaFluor647 or APC.Incubation occurred overnight at room temperature in 1xPBS. For antibodies that werenot directly conjugated, two primaries were added, one made in rabbits and one in mice.In that case, after washing with 1xPBS, secondary antibodies were applied and incubatedfor 15 min: a mix of for example: horse Anti-Rabbit IgG Antibody (H+L), DyLight® 594(DI-1094, Vector Laboratories, Newark, CA, USA) and horse Anti-Mouse IgG Antibody(H+L), DyLight® 488 (DI-2488, Vector Laboratories, Newark, CA, USA), both 1:400 dilutedin 1xPBS with 5%BSA. After washing with 1xPBS, sections were counterstained with2µM Hoechst dye for 10 min. After washing, slides were provisionally mounted withVECTASHIELD® Antifade Mounting Medium (H-1000, Vector Laboratories, Newark, CA,USA) and imaged using a Leica SP5 confocal microscope (Leica Microsystems, Deerfield, IL,USA) with a motorized stage and the 63× (Leica 1.4-0.6 HCX PL APO, oil) or 20x objective(Leica 20×/0.7 HC PL APO). After imaging, slides were unmounted and washed in 1xPBS.Int. J. Mol. Sci. 2025, 26, 3144 21 of 26Antibodies were removed by microwaving slides in 1xTE buffer. Boiling the slides wasrepeated five more times over the next 40 min and then the slides were cooled down toroom temperature. Additionally, a peroxide-light bleaching step was included especially forstains using directly conjugated antibodies: sections were incubated between 10 and 45 minin a mix of 7.5 mL PBS, 0.24 mL 1M NaOH, 1.35 mL 30%H2O2 and exposed to maximumlight intensity from two Aliyeah Xz-06 22600Lux LED light sources. Photobleaching timeswere dependent on signal intensity and fluorochrome. Then, the next round of primaryantibodies was applied. The same areas from the initial imaging session were imaged,analyzed, and quantified using Leica LASX software version 3.7 (Leica Microsystems,Deerfield, IL, USA). To produce overlay images from all the cycles of the same tissue area,Leica files were exported as TIFFs and assembled in Adobe Photoshop version 26 (Adobe,San Jose, CA, USA). Images were analyzed using Miltenyi MACSiQView 1.2.2 and 1.3software (Miltenyi Biotec, Gaithersburg, MD, USA). In manual analysis, we relied on visualdetermination of basal cell identity due to high resolution and high magnification of images.Additionally, we used basal cell markers as guides of basal cell identity. In Figure 2, weused this “manual” determination of basalness to compare basal and suparabasal geneexpression. For scatterplots, we determined the position of the gates by determining thelowest level of expression of clearly positive cells within the images.The manually multiplex images were also used to determine the gene expression andnumber of delaminating cells. A delaminating cell was defined as a cell that has still aconnection to the basal membrane but has left the group of basal cells and moved upwardsout of the basal plain.4.4. Automated Multiplex Immunofluorescence Staining Using the Miltenyi MACSima PlatformFor antigen retrieval, tissue slides were incubated for 20 min at 98 ◦C in 1xTECpH9 buffer (made from 10x TEC: 2.5 g Trizma Base (Sigma-Aldrich/Merck, Burling-ton, MD, USA, # 93362), 5.75 g EDTA Disodium salt dihydrate (Sigma-Aldrich/Merck,Burlington, MD, USA, #E5134), 3.2 g Sodium citrate tribasic dihydrate (Sigma-Aldrich/Merck,Burlington, MD, USA, #1064480500), pH adjusted with NaOH. After cooling down, theslides were mounted onto a MACSwell Imaging Frame and loaded onto the MACSimastaining and imaging platform (all Miltenyi Biotec, Gaithersburg, MD, USA). Followingthe manufacturer’s recommendations, Miltenyi antibodies were diluted in MACSima Run-ning buffer. After preprocessing of image data, images were analyzed using MiltenyiMACSiQView 1.2.2 and 1.3 software (Miltenyi Biotec, Gaithersburg, MD, USA).4.5. Proximity Ligation Assays (PLA)The PLA was performed on a tissue microarray (TMA) slide (BBS02011 from TissueAr-ray.Com LLC, Derwood, MD, USA using the Duolink® Proximity Ligation Assay fromMilliporeSigma, Burlington, MA, USA according to the manufacturer’s recommendationswith the following changes: Primary antibodies (MECP2 1:100, clone D4F3, #3456, CellSignaling Technology, Danvers, MA, USA) and XPC 1:100; D-10, sc-74410, Santa CruzBiotechnology, Dallas, TX, USA) were incubated overnight at room temperature. Secondaryantibodies (Duolink in situ PLA probe anti rabbit plus and Duolink in situ PLA probe antimouse minus) were incubated for 2 h at 37 ◦C. The ligation reaction was extended to 1 hand the amplification reaction to 110 min. The TMA slide was imaged using a Leica Sp5confocal microscope using a 20× objective. After imaging, the cover glass was removed inwater and the TMA incubated with anti-rabbit IgG Alexa647 (A21245 Invitrogen, Carlsbad,CA, USA) and anti-mouse IgG Fluorescein (FI-2000 Vector Laboratories, Newark, CA, USA)to detect and visualize MECP2 and XPC, respectively. After overnight incubation, the TMAwas imaged again at the same location that the PLA signal was imaged. After washing offInt. J. Mol. Sci. 2025, 26, 3144 22 of 26the cover glass and photobleaching the exiting fluorescent signals, a KRT10/13 antibody(clone REA1138 FITC Miltenyi Biotec, Gaithersburg, MD, USA) and a Ki-67 antibody (cloneD3B5 AlexaFluor647, Cell Signaling Technology, Danvers, MA, USA) were added overnightand imaged again the next day to aid in cell clustering in downstream UMAP analyses.After cooking the slide in the microwave and photobleaching for 25 min, the TMA slidewas incubated one last time with KRT14 (REA1145 FITC Miltenyi Biotec, Gaithersburg,MD, USA), VIM (D21H3 PE Cell Signaling Technology, Danvers, MA, USA), and SLC3A2(e-5 sc376815 AlexaFluor647, Santa Cruz Biotechnology, Dallas, TX, USA). The images werealigned in Photoshop using the DAPI stains of each day. The resulting TIFF images wereimported into MACSiQView version 1.3 (Miltenyi Biotec, Gaithersburg, MD, USA) forfurther analysis and quantification. Additional PLA controls were performed ahead of theTMA stain using two Ki-67 antibodies as positive control (see Supplementary Figure S4). Afterthe end of staining and imaging, a standard hematoxylin and eosin stain was performedand imaged.4.6. TissuesAnimal tissues and their collection have previously been described [25]. Human tissuesamples were obtained from the Cooperative Human Tissue Network (CHTN). Additionalhuman samples were obtained from TissueArray.Com LLC, Derwood, MD, USA in form ofhuman tissue microarrays (TMAs: ES809, BBS02011).4.7. Analysis of scRNAseq DataDatasets included in the study are from published work [82,86–92]. For the gene setsspecifically expressed, see Supplementary Table S6.4.8. Statistical AnalysisStatistical analyses and graphs were generated using Prism Software (version 10.2.3,Graphpad, Boston, MA, USA). Statistical significance was determined using Student's t-test,with p-values ≤ 0.05 denoted by an asterisk (*). For Figure 1, Figure 3, and Figure 5, dataare represented as the median with the 25th and 75th percentiles (interquartile range, IQR),and the whiskers extend from the minimum to the maximum values. In Figure 7, outliersare shown; here, the whiskers extend to the minimum and maximum values within 1.5times the IQR, with outliers plotted as individual points.Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26073144/s1.Author Contributions: Conceptualization, T.A. and C.D.A.; methodology, T.A.; validation, Y.P.;formal analysis, A.H., K.B., R.P., A.J. and R.E.G.-R.; investigation, A.H., K.B., R.P., A.J., L.H., R.E.G.-R.and T.A.; resources, L.H., C.D.A. and T.A.; data curation, A.H., K.B., R.P., A.J., L.H., R.E.G.-R. andT.A.; writing—original draft preparation, A.H. and T.A.; writing—review and editing, A.H., K.B.,R.P., A.J., L.H., R.E.G.-R., T.A and C.D.A.; visualization, A.H., K.B., R.P., A.J. and T.A.; supervision,C.D.A. and T.A.; project administration, T.A. and Y.P. evaluate the grade of dysplastic lesions in thetissue microarrays. All authors have read and agreed to the published version of the manuscript.Funding: This research received no external funding.Institutional Review Board Statement: The study was conducted in accordance with the Declarationof Helsinki and approved by the Institutional Review Board of The University of Central Floridawhich determined in IRB ID: SBE-16-12646 that the research activity associated with this manuscriptis not human research as defined by DHHS regulations at 45 CFR 46. The animal material used withinthis manuscript has been reviewed by several IACUCs and deemed not animal research.Informed Consent Statement: Not applicable.Int. J. Mol. Sci. 2025, 26, 3144 23 of 26Data Availability Statement: Upon request, we will share the protein expression data from ourmultiplex expression analyses in form of Excel sheets, .csv or .fcs files.Acknowledgments: We would like to thank Daniel Zhang for help with stainings, imaging andimage analysis. 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Quantitative ClonalAnalysis and Single-Cell Transcriptomics Reveal Division Kinetics, Hierarchy, and Fate of Oral Epithelial Progenitor Cells. CellStem Cell 2019, 24, 183–192.e188. [CrossRef] [PubMed]Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individualauthor(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.
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squamous epithelial biology
esophageal stem cells
spatial biology
multiplex immunofluorescence
comparative biology
MECP2
XPC
Human-Specific Organization of Proliferation and Stemness in Squamous Epithelia : A Comparative Study to Elucidate Differences in Stem Cell Organization
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