Single-cell analysis of intestinal epithelial organoids reveals aging-associated differentiation delay conserved between in vivo and ex vivo

Single-cell analysis of intestinal epithelial organoids reveals aging-associated differentiation delay conserved between in vivo and ex vivo | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Single-cell analysis of intestinal epithelial organoids reveals aging-associated differentiation delay conserved between in vivo and ex vivo Keisho Hasegawa, Masaya Kimura, Kensho Toyoshima, Riko Yokoyama, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8093838/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Intestinal epithelial stem cells (ISCs) are essential for maintaining the structural and functional integrity of the gut by continuously replenishing the intestinal epithelium. However, the effects of aging on the identity and function of ISCs and their progeny remain poorly understood. In this study, we performed single-cell RNA sequencing (scRNA-seq) to analyze the cellular composition and transcriptional landscape of intestinal epithelial organoids derived from young and aged mice. To complement and validate our organoid findings, we also examined publicly available scRNA-seq datasets from intestinal epithelial tissues of mice across different age groups. Our analysis revealed that ISCs from aged mice exhibited impaired differentiation trajectories, with a pronounced delay in the transition to transit-amplifying (TA) cells and later stages. Remarkably, the same differentiation delay and transcriptional changes were observed in native intestinal tissues, indicating that organoids can serve as faithful models for studying aging of stem cell-derived epithelial tissue. Additionally, a delay in differentiation was also observed in organoids derived from aged mice, manifested as a delayed onset of budding structures. Our results presented fundamental insights into the aging of ISCs conserved between in vivo and ex vivo and established an ex vivo platform for evaluating therapeutic interventions targeting intestinal regeneration in aging. Biological sciences/Cell biology Biological sciences/Developmental biology Biological sciences/Stem cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The intestinal epithelium is one of the most rapidly renewing tissues in the body, and this renewal is sustained by intestinal epithelial stem cells (ISCs) located at the base of the crypts (Rodriguez-Colman et al., 2017). These cells give rise to proliferative transit-amplifying (TA) cells, which further differentiate into various specialized epithelial lineages, including enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. The precise regulation of ISC self-renewal and differentiation is essential for maintaining intestinal homeostasis. Aging is associated with a progressive decline in the regenerative capacity of many tissues, including the intestine. Previous studies have suggested that aging leads to reduced stem cell function, altered lineage allocation, and increased susceptibility to inflammation and disease (Oh, Lee, & Wagers, 2014) . In the intestinal epithelium, aging can alter crypt architecture, reduce Lgr5+ ISC abundance, and impair Wnt signaling (Nalapareddy et al., 2017; Pentinmikko et al., 2019). Additionally, aged ISCs exhibit impaired responses to injury and diminished organoid-forming capacity (Igarashi et al., 2019). Transcriptional profiling studies have further revealed age-associated changes in stem cell identity and epigenetic regulation (Choi et al., 2023; Moor et al., 2017) . Despite these insights, the specific cellular and molecular mechanisms through which aging impairs ISC differentiation remain unclear. In particular, the early transition from stem cells to TA cells is critical for epithelial turnover but has not been well-characterized in the context of aging. To address these questions, we performed single-cell RNA sequencing (scRNA-seq) to profile organoids derived from intestinal crypts of young and aged mice. Organoids serve as powerful ex vivo models that recapitulate key features of the intestinal epithelium, including cell-type diversity and stem cell hierarchies. We complemented our analysis with scRNA-seq data from native intestinal tissues to assess the physiological relevance of the observed organoid changes. Our findings revealed age-associated delays in ISC differentiation, particularly at early steps, and established a framework for using organoids to model epithelial aging. Materials and Methods Establishment of intestinal epithelial organoids Isolation and dissociation of stem cells from the normal intestinal epithelium of wild-type C57BL/6 mice were performed as previously described (Uchida et al., 2019) . Isolated epithelial cells were embedded in Matrigel on ice (growth factor-reduced, phenol red-free; Corning, Corning, NY, USA) and seeded in 48-well plates. The Matrigel was polymerized for 10 min at 37 °C, and overlaid with 250 μL/well basal culture medium (advanced Dulbecco’s modified Eagle medium/F12 supplemented with penicillin/streptomycin, 10 mmol/L HEPES, Glutamax, 1 × N2, 1 × B27 [all from Thermo Fisher Scientific, Waltham, MA, USA], and 1 mmol/L N-acetylcysteine [Sigma-Aldrich, St. Louis, MO, USA]) containing the following optimized growth factor combinations: murine epidermal growth factor (Thermo Fisher Scientific), recombinant murine noggin (Peprotech, Rocky Hill, New Jersey, USA), Y-27632 (Selleck, Yokohama, Kanagawa, Japan), and R-spondin 1. In this study, mice older than 50 weeks were considered aged, and the establishment of intestinal epithelial organoids was deemed successful when cultures were maintained for over five passages. scRNA-seq analysis For scRNA-seq analysis, we used organoid-derived intestinal epithelial cells from 21-week-old (young) and 112-week-old (aged) mice. Organoids were dissociated into single cells using TrypLE (Thermo Fisher Scientific), filtered through a cell strainer, and resuspended in 2% BSA/PBS. scRNA-seq was performed using the Chromium Next GEM Single Cell 3’ Kit v3.1 with Dual Index (10x Genomics, Pleasanton, CA, USA), following the manufacturer’s protocol. Briefly, single-cell suspensions were loaded onto the Chromium Controller to generate Gel Bead-in-Emulsions (GEMs), and reverse transcription was conducted within droplets. Following complementary DNA (cDNA) amplification, three-prime gene expression libraries with dual indexing were constructed. cDNA amplicons were enzymatically fragmented and size-selected to achieve optimal insert sizes. Subsequently, end repair, A-tailing, adaptor ligation, and PCR amplification were performed to incorporate the P5 and P7 flow cell adaptors, i7 and i5 sample indices, and TruSeq Read 2 primer sequence. The resulting libraries were sequenced on an Illumina NovaSeq 6000 platform with the following configuration: paired-end, dual indexing, Read 1: 28 cycles, Read 2: 90 cycles, Index read i7: 10 cycles, and Index read i5: 10 cycles, targeting a sequencing depth of 2,000 read pairs per cell. Base calls were converted to FASTQ files and aligned to the mouse reference genome using Cell Ranger to generate the gene-cell count matrix. Downstream data processing and analysis were performed using the Seurat R package on R. Low-quality cells, including those with 10% mitochondrial gene expression, or identified as potential doublets, were filtered out. After quality control, data were log-normalized and scaled to correct for technical variations, such as library size differences. For dimensionality reduction, principal component analysis (PCA) was performed using Seurat’s RunPCA function, and the number of significant principal components was selected based on the ElbowPlot. The data were further visualized using Uniform Manifold Approximation and Projection (UMAP). Cluster identification was conducted, and cell-type annotation was based on the expression of canonical marker genes. We also analyzed publicly available scRNA-seq datasets of intestinal epithelial tissues from young (5-month-old) and aged (24-month-old) mice, which are deposited in the Gene Expression Omnibus (GEO) database under accession number GSE210669 (Choi et al., 2023) . Pseudotime analysis We utilized pseudotime analysis to reconstruct the differentiation trajectory of stem cells and to visualize gene expression changes associated with aging. Pseudotime analysis was performed using the Monocle 3 R package to infer the dynamic cellular trajectories during intestinal epithelial differentiation. All analyses were performed using R version 4.3.3, and statistical significance was set at p < 0.05. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis For passaged and cultured organoids, Cell Recovery Solution (Corning) was added to each well, and the plate was kept on ice for 1 h. Cells were centrifuged for 2 min, the supernatant was removed, and total RNA was extracted using QIAshredder (QIAGEN, Hilden, Germany) and the RNeasy Mini Kit (QIAGEN), according to the manufacturer’s instructions. The purified RNA was dissolved in 30 µL of RNase-free water and quantified using a NanoDrop 2000c spectrophotometer (Thermo Scientific). cDNA was synthesized from the extracted RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) with RNase inhibitor, following the manufacturer's protocol. Reverse transcription was performed using a Mastercycler nexus gradient (Eppendorf, Hamburg, Germany). Relative cDNA quantification was performed using the Pfaffl method. qPCR was carried out with PowerTrack™ SYBR Green Master Mix for qPCR (Thermo Fisher Scientific) in MicroAmp™ Optical 96-Well Reaction Plates (Thermo Fisher Scientific), and fluorescence was detected using the CFX Connect™ Real-Time PCR System (Bio-Rad laboratories, Hercules, CA, USA). Primer sequences are listed in Supplementary Table 1. Evaluation of temporal morphological changes of intestinal epithelial organoids Images of organoids were acquired on days 3 to 6 after passaging. Image analysis was performed under blinded conditions. The number of spherical organoids (defined as organoids without any budding structures; organoids showing at least one bud were excluded) was visually counted in each image. This process was performed three times independently, and the images were subsequently unblinded to confirm their classification as young or old organoids. Finally, the mean number of spherical organoids per image was calculated. Statistical Analysis Statistical analyses were carried out using R. Comparisons between two groups were assessed using a two-tailed unpaired t-test. Welch's t-test was applied when variances were unequal and Student's t-test was used otherwise. For time-series data, differences between two groups were evaluated using a linear mixed-effects model with the Satterthwaite-adjusted F-test. Spearman's rank correlation was assessed using a t-distribution-based significance test. For all analyses, p values < 0.05 were considered statistically significant. For the graph in Figure 7, the regression line and its 95% confidence interval were generated using a Pearson correlation (linear regression), while the statistical significance of the association was assessed using Spearman's rank correlation to ensure robustness against non-linear relationships and potential outliers. Ethics Statement This work was approved by the Animal Experiments and Experimental Animal Welfare Committee of Keio University (Approved No. A2022-105), and a proof/certificate of approval is available upon request. Results Establishment of i ntestinal e pithelial o rganoids and scRNA-seq a nalysis We established organoids using intestinal epithelial tissues obtained from mice at various ages. Representative images of a single stem cell expanding into organoids derived from intestinal epithelia of mice are shown in Figure 1A. Intestinal epithelial organoids derived from young mice demonstrated vigorous growth and expanded through budding structures resembling crypts that harbor stem cells. In contrast, organoids derived from the intestinal epithelium of aged mice demonstrated a lower establishment efficiency. Moreover, once established, these organoids exhibited reduced proliferative capacity and limited budding formation (Figure 1A). Figure 1B shows the UMAP results from single-cell analysis of intestinal epithelial organoids derived from both young and aged mice. The analysis revealed that the organoids were classified into ten distinct clusters, confirming that they contained all major intestinal epithelial cell types, including stem cells (ISCs), TA cells, absorptive enterocyte precursors (AEPs), secretory progenitor cells (SPCs), enterocytes, enteroendocrine cells (EECs), Paneth cells, goblet cells, and tuft cells. Figure 1C shows violin plots of the expression levels of marker genes for each cluster in the intestinal epithelial organoids. The marker genes used for identifying each cell type were as follows: Lgr5 and Olfm4 for stem cells (ISCs), Mki67 for TA cells, Hes1 for AEPs, Alpi, Fabp1 , Slc5a1 , and Slc2a2 for enterocytes, Chga for EECs, Lyz1 for Paneth cells and goblet cells, Muc2 for goblet cells, and Dclk1 for tuft cells. Pseudotime analysis using intestinal epithelial organoids derived from young and aged mice Next, we performed pseudotime analysis using intestinal epithelial organoids derived from young and aged mice. Pseudotime analysis is a computational method used in scRNA-seq studies to infer the temporal order of cells along a biological process, such as differentiation or development. It does not measure actual time but instead estimates a trajectory that reflects how cells transition from one state to another based on their gene expression profiles. Figure 2A shows the results of pseudotime analysis of intestinal epithelial organoids derived from young and aged mice. The arrows indicate the starting point of differentiation from stem cells. As shown in Figure 2A, organoids derived from young mice contain fewer early-stage stem cells but a higher proportion of cells at later stages of differentiation compared to those from aged mice. In contrast, organoids derived from aged mice have a larger number of early-stage stem cells and fewer cells at later stages of differentiation. Therefore, we analyzed the relationship between pseudotime differentiation and the proportion of each cell type in intestinal epithelial organoids derived from young and aged mice. As shown in Figure 2B, all examined cell types—stem cells, TA cells, enterocytes, and EECs—in intestinal epithelial organoids derived from aged mice exhibited delayed differentiation compared to those from young mice. The pseudotime analysis revealed significant differences in differentiation trajectories between young and aged mice across all examined cell types: Stem cells: Mean pseudotime was 0.0875 in young and 0.2232 in aged mice (p < 0.001). Transit-amplifying (TA) cells: Mean pseudotime was 0.2285 in young and 0.5103 in aged mice (p < 0.001). Enterocytes: Mean pseudotime was 0.3734 in young and 0.6901 in aged mice (p < 0.001). Enteroendocrine cells (EECs): Mean pseudotime was 0.6341 in young and 0.9657 in aged mice (p < 0.001). These results indicate a consistent delay in cellular differentiation in the aged intestinal epithelium. scRNA-seq and pseudotime analyses using cells derived from intestinal epithelial tissues of young and aged mice Next, we performed a similar analysis using publicly available scRNA-seq data of intestinal epithelial cells from young and aged mice published by Choi et al. (Choi et al., 2023) and compared the results with those obtained from our intestinal epithelial organoids. Figure 3A shows the UMAP results from single-cell analysis of cells derived from intestinal epithelial tissues of young and aged mice. Similar to our organoid data, the single-cell analysis of tissue-derived cells revealed ten distinct clusters, confirming the presence of all major intestinal epithelial cell types, including stem cells (ISCs), TA cells, AEPs, SPCs, enterocytes, EECs, Paneth cells, goblet cells, and tuft cells. Figure 3B shows violin plots of the expression levels of marker genes for each cluster in cells derived from intestinal epithelial tissue. The marker genes used to identify each cell type were as follows: Lgr5 and Olfm4 for stem cells (ISCs), Mki67 for TA cells, Hes1 for AEPs, Alpi, Fabp1 , Slc5a1 , and Slc2a2 for enterocytes, Chga for EECs, Lyz1 for Paneth cells and goblet cells, Muc2 for goblet cells, and Dclk1 for tuft cells. Figure 4A shows the results of pseudotime analysis using cells derived from the intestinal epithelial tissue of young and aged mice. The arrow indicates the starting point of differentiation from stem cells. In the tissue derived from young mice, a greater number of cells were observed at later stages of differentiation. In contrast, the tissue from aged mice exhibited fewer cells at the later stages, suggesting impaired or delayed differentiation with aging. To further assess the effects of aging on cellular differentiation in intestinal epithelial tissue, we conducted pseudotime analysis using cells derived from young and aged mice. As shown in Figure 4B, all examined cell types—stem cells, transit-amplifying (TA) cells, and tuft cells—exhibited significantly higher pseudotime values in aged mice compared to young mice, indicating delayed differentiation with aging: Stem cells, the mean pseudotime was 0.1059 in young mice and 0.1766 in aged mice (p < 0.001). TA cells, the mean pseudotime was 0.1445 in young mice and 0.4983 in aged mice (p < 0.001). Tuft cells, the mean pseudotime was 0.7164 in young mice and 0.7714 in aged mice (p < 0.001). These findings further supported the observation that aging is associated with a consistent delay in intestinal epithelial cell differentiation in both tissues and organoid models. Temporal morphological and marker gene expression changes in intestinal epithelial organoids Delayed differentiation also is expected to affect budding formation, a characteristic feature of intestinal epithelial organoids. Therefore, to more definitively confirm the possibility of delayed differentiation suggested by the scRNA-seq results, we examined how it manifests in organoids ex vivo . Intestinal epithelial organoids are spherical immediately after passage, but gradually acquire the budding structures characteristic of intestinal epithelial organoids. After passage, we evaluated changes in organoid morphology by observing the growth of intestinal epithelial organoids over time and counting the number of spherical organoids relative to the total organoid area within the field of view. Figure 5A shows the counting process of spherical organoids and Figure 5B shows its temporal morphological changes. Figure 5B indicates that organoids with budding structures appeared earlier in young mice than in aged mice, while spherical organoids persisted longer in aged mice (p=0.0363). Budding structures are known to form with the appearance of Paneth cells, one of the final differentiated cell types (Sato et al., 2009; Sato et al. 2011; Serra et al., 2019), indicating that budding formation commences upon complete differentiation. Thus, delayed budding formation indicates delayed differentiation progression. To investigate changes in the expression of marker genes associated with morphological changes in intestinal epithelial organoids, we performed RNA extraction from organoids at days 3 to 6 after passage. RT-qPCR was performed for the marker genes and correlations between their expression levels and organoid morphology were calculated (Figure 6, Figure 7). As shown in Figure 6, expression of Mki67 , a marker gene of TA cells, Muc2 , a marker gene of goblet cells, and Chga , a marker gene of EECs, was significantly higher in aged mice compared to young mice. Conversely, expression of Lyz1 , which is a marker gene of Paneth cells, was significantly higher in young mice compared to aged mice. Furthermore, correlations between the expressions of the marker genes and the morphological changes in intestinal epithelial organoids were particularly strong for Lgr5 , Slc2a2 , Lyz1 , and Dclk1 (rho≧0.7 or rho≦-0.7). These results were consistent with previous research asserting that Paneth cell activity is necessary for budding formation in intestinal epithelial organoids (Sato et al., 2009; Sato et al., 2011; Serra et al., 2019). Discussion This study provided comprehensive single-cell resolution insights into the effects of aging on intestinal epithelial stem cell differentiation. We demonstrated that aging impaired the early transition of ISCs to TA cells, leading to altered epithelial renewal. Importantly, these findings were consistent across both organoid models and native tissues, supporting the robustness and physiological relevance of the observations. Additionally, intestinal epithelial organoids exhibit an age-associated decline in differentiation capacity and function, manifested as delayed formation of budding structures. The results from pseudotime analysis of scRNA-seq data from intestinal epithelial tissue and intestinal epithelial organoids suggested that differentiation from ISCs to TA cells and later stages may be delayed with aging, both in vivo and ex vivo . Furthermore, although the intestinal epithelial tissue was derived from a hybrid mouse strain, similar results were obtained with intestinal epithelial organoids derived from C57BL/6J mice. This suggested that the observed aging changes occur to some extent regardless of genetic diversity. Lyz1 expression, a Paneth cell marker, showed a strong inverse correlation with the number of spherical organoids, and its increased expression was significantly associated with a decrease in the number of spherical organoids. This indicated that the activity level of Paneth cells, specifically those expressing Lyz1 (where activity level refers to either higher cell counts or stronger cellular function), is strongly associated with budding formation in intestinal epithelial organoids. This aligned with previous research asserting that Paneth cell activity is necessary for budding formation in intestinal epithelial organoids (Sato et al., 2011; Sato et al., 2009; Serra et al., 2019) . The number of spherical organoids and RT-qPCR results suggested that age-related decline in Paneth cell function may delay differentiation from stem cells to TA cells and later stages. While Paneth cells exist in crypts alongside stem cells and are known to regulate stem cell differentiation by modulating Wnt and Notch signaling intensity, it is also known that while their number increases with aging, their function declines (Wallaeys, Garcia-Gonzalez, & Libert, 2023). Considering that pseudotime analysis in scRNA-seq assigns pseudotime to each cell in mature intestinal epithelial tissue and intestinal epithelial organoids, it is necessary to consider that dysfunctional Paneth cells may already exist around stem cells in these tissues and organoids, potentially affecting the stem cell transcriptome. Integrating our results with previous studies suggests that age-related changes in cell-cell interactions, particularly a decline in Paneth cell function, contribute to delayed differentiation from stem cells to TA cells and later stages. Our pseudotime analysis with both organoid and in vivo tissue data complements and extends prior research, offering a dynamic and comparative view of age-related stem cell dysfunction. By excluding systemic influences, organoids serve as ideal models to dissect epithelial-intrinsic aging mechanisms and to screen rejuvenation strategies. Intestinal epithelial organoids express the decline in differentiation potential and function into Paneth cells, which also occurs in intestinal epithelial tissue with aging, with the accompanying reduction in stem cell differentiation potential manifested as delayed formation of budding structures. In other words, the differentiation capacity of intestinal epithelial organoids reflects age-related changes in intestinal epithelial tissue differentiation. By focusing on delayed budding structure formation and differentiation of cell marker genes, it may be possible to conduct aging research ex vivo . In the broader context of aging biology, our findings underscore the critical role of stem cell dynamics in tissue homeostasis and age-related pathologies. Understanding how aging disrupts ISC behavior may inform strategies to enhance intestinal regeneration and resilience in the elderly. Conclusion Intestinal epithelial organoids faithfully modeled age-related impairments in stem cell differentiation. Aging delayed the transition of ISCs to TA cells and later stages, reflecting a key vulnerability in epithelial renewal. Our study established a foundation for future research into intestinal aging and provided a tractable system for therapeutic development. Moreover, our study suggested that organoids have potential as models of stem cell-derived tissue aging. Declarations Acknowledgements We thank the Animal Facility staff for their daily care of the laboratory animals. Also, we would like to thank Editage (www.editage.jp) for English language editing. Funding This work was supported by JSPS KAKENHI (Grant Numbers JP24KK0207 and JP24K03285) grants awarded to Y.S. Author’s Contributions K.H., Masaya K., Masaki K., J.M., J.N., and Y.S. designed the experiments and supervised the work. K.T., R.Y., and T.M. performed data processing. K.H. and Masaya K. performed the experiments and statistical analysis. K.H., Masaya K., and Y.S. generated the figures. K.H., T.I., and Y.S. wrote and edited the manuscript. All authors read and approved the final manuscript. Conflicts of Interest All the authors declare that there are no conflicts of interest. References Choi, J., Houston, M., Wang, R., Ye, K., Li, W., Zhang, X., . . . Augenlicht, L. H. (2023). Intestinal stem cell aging at single-cell resolution: Transcriptional perturbations alter cell developmental trajectory reversed by gerotherapeutics. Aging Cell, 22 (5), e13802. doi:10.1111/acel.13802 Igarashi, M., Miura, M., Williams, E., Jaksch, F., Kadowaki, T., Yamauchi, T., & Guarente, L. (2019). NAD(+) supplementation rejuvenates aged gut adult stem cells. Aging Cell, 18 (3), e12935. doi:10.1111/acel.12935 Moor, A. E., Golan, M., Massasa, E. E., Lemze, D., Weizman, T., Shenhav, R., . . . Itzkovitz, S. (2017). Global mRNA polarization regulates translation efficiency in the intestinal epithelium. Science, 357 , 1299-1303. doi:10.1126/science.aan2399 Nalapareddy, K., Nattamai, K. J., Kumar, R. S., Karns, R., Wikenheiser-Brokamp, K. A., Sampson, L. L., . . . Geiger, H. (2017). Canonical Wnt Signaling Ameliorates Aging of Intestinal Stem Cells. Cell Reports, 18 (11), 2608-2621. doi:10.1016/j.celrep.2017.02.056 Oh, J., Lee, Y. D., & Wagers, A. J. (2014). Stem cell aging: mechanisms, regulators and therapeutic opportunities. Nature Medicine, 20 (8), 870-880. doi:10.1038/nm.3651 Pentinmikko, N., Iqbal, S., Mana, M., Andersson, S., Cognetta, A. B., 3rd, Suciu, R. M., . . . Katajisto, P. (2019). Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium. Nature, 571 (7765), 398-402. doi:10.1038/s41586-019-1383-0 Rodriguez-Colman, M. J., Schewe, M., Meerlo, M., Stigter, E., Gerrits, J., Pras-Raves, M., . . . Burgering, B. M. (2017). Interplay between metabolic identities in the intestinal crypt supports stem cell function. Nature, 543 (7645), 424-427. doi:10.1038/nature21673 Sato, T., van Es, J. H., Snippert, H. J., Stange, D. E., Vries, R. G., van den Born, M., . . . Clevers, H. (2011). Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature, 469 (7330), 415-418. doi:10.1038/nature09637 Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., . . . Clevers, H. (2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature, 459 (7244), 262-265. doi:10.1038/nature07935 Serra, D., Mayr, U., Boni, A., Lukonin, I., Rempfler, M., Challet Meylan, L., . . . Liberali, P. (2019). Self-organization and symmetry breaking in intestinal organoid development. Nature, 569 (7754), 66-72. doi:10.1038/s41586-019-1146-y Uchida, R., Saito, Y., Nogami, K., Kajiyama, Y., Suzuki, Y., Kawase, Y., . . . Saito, H. (2019). Erratum: Publisher Correction: Epigenetic silencing of Lgr5 induces senescence of intestinal epithelial organoids during the process of aging. NPJ Aging Mech Dis, 5 , 5. doi:10.1038/s41514-019-0035-9 Wallaeys, C., Garcia-Gonzalez, N., & Libert, C. (2023). Paneth cells as the cornerstones of intestinal and organismal health: a primer. EMBO Molecular Medicine, 15 (2), e16427. doi:10.15252/emmm.202216427 Additional Declarations No competing interests reported. Supplementary Files 251109AgingPaperSupplementaryTable1.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 04 Dec, 2025 Reviews received at journal 04 Dec, 2025 Reviews received at journal 03 Dec, 2025 Reviews received at journal 29 Nov, 2025 Reviewers agreed at journal 26 Nov, 2025 Reviewers agreed at journal 25 Nov, 2025 Reviewers agreed at journal 24 Nov, 2025 Reviewers invited by journal 24 Nov, 2025 Editor assigned by journal 19 Nov, 2025 Submission checks completed at journal 17 Nov, 2025 First submitted to journal 12 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8093838","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":551427442,"identity":"e4ec3d78-5f83-433a-a98f-ae2a5dfe46e9","order_by":0,"name":"Keisho Hasegawa","email":"","orcid":"","institution":"Keio University Faculty of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Keisho","middleName":"","lastName":"Hasegawa","suffix":""},{"id":551427443,"identity":"6a866332-d17b-43e7-83f3-4ae3e04664ba","order_by":1,"name":"Masaya Kimura","email":"","orcid":"","institution":"Keio University Faculty of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Masaya","middleName":"","lastName":"Kimura","suffix":""},{"id":551427446,"identity":"9f8b8096-2899-48b3-889f-d74a9da20f28","order_by":2,"name":"Kensho Toyoshima","email":"","orcid":"","institution":"Keio University Faculty of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Kensho","middleName":"","lastName":"Toyoshima","suffix":""},{"id":551427447,"identity":"5bf3879d-3fb8-4452-a80e-ee9baf20cb50","order_by":3,"name":"Riko Yokoyama","email":"","orcid":"","institution":"Keio University Faculty of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Riko","middleName":"","lastName":"Yokoyama","suffix":""},{"id":551427449,"identity":"e3158184-5965-4b74-8e6a-daca95571884","order_by":4,"name":"Toshihide Muramatsu","email":"","orcid":"","institution":"Keio University Faculty of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Toshihide","middleName":"","lastName":"Muramatsu","suffix":""},{"id":551427451,"identity":"9c04e230-2033-4201-abce-7875e68d8ae8","order_by":5,"name":"Takaaki Ishikawa","email":"","orcid":"","institution":"Keio University Faculty of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Takaaki","middleName":"","lastName":"Ishikawa","suffix":""},{"id":551427452,"identity":"fc354937-9147-4fee-92e1-d7eacd4b8e3f","order_by":6,"name":"Masaki Kimura","email":"","orcid":"","institution":"Keio University Faculty of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Masaki","middleName":"","lastName":"Kimura","suffix":""},{"id":551427454,"identity":"39b025c3-ed4e-40b4-8bd8-7bd83293a05c","order_by":7,"name":"Juntaro Matsuzaki","email":"","orcid":"","institution":"Keio University Faculty of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Juntaro","middleName":"","lastName":"Matsuzaki","suffix":""},{"id":551427456,"identity":"237d5ccb-f4ef-468b-aa1f-a191511edceb","order_by":8,"name":"Jun Nakayama","email":"","orcid":"","institution":"Osaka International Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Nakayama","suffix":""},{"id":551427457,"identity":"3d1fb500-b176-4db2-8aaf-84129f35ccbb","order_by":9,"name":"Yoshimasa Saito","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYJCCAwwMNgYMzECWBNFaDjCkkagFaM1hA+IdZXD+8MPDHyrOG/OzMx9gsNxBjJYbaQYHDpy5bSbZzJbAIHmGKC0MBgcOtt22MTjMY8Ag2UaUw45/AGo5Z2NPvJYDOSBbDpgZMBOrRfJGTsGBM2eSjSUOsyUcIMovfOePb/5QUWFn2N9/+OBjSWJCTOEAEuewZAMRWuSRFTF+JEbLKBgFo2AUjDgAAHIJO/HJRGRoAAAAAElFTkSuQmCC","orcid":"","institution":"Keio University Faculty of Pharmacy","correspondingAuthor":true,"prefix":"","firstName":"Yoshimasa","middleName":"","lastName":"Saito","suffix":""}],"badges":[],"createdAt":"2025-11-12 08:23:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8093838/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8093838/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96987540,"identity":"e7b21c5c-73b1-422c-918a-0dcc3f13dba5","added_by":"auto","created_at":"2025-11-28 10:33:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":967027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003escRNA-seq analysis of intestinal epithelial organoids:\u003c/strong\u003e (A) Representative images of intestinal epithelial organoids derived from young and aged mice. (B) UMAP plot showing the clustering of cells from scRNA-seq analysis of intestinal epithelial organoids. “Total” indicates the combined dataset of young and aged samples. Cluster labels indicate cell types. TA cells, transit-amplifying cells; Stem-TA cells indicate cells within this cluster have both expression of stem-cell-marker genes and TA-cell-marker genes; AEPs, absorptive epithelial progenitor cells; SPCs, secretory epithelial progenitor cells; EECs, enteroendocrine cells. (C)The marker genes used for identifying each cell type are as follows: \u003cem\u003eLgr5\u003c/em\u003e and\u003cem\u003e Olfm4\u003c/em\u003e for stem cells (ISCs), \u003cem\u003eMki67\u003c/em\u003e for TA cells, \u003cem\u003eHes1\u003c/em\u003e for \u003cem\u003eAEPs\u003c/em\u003e, \u003cem\u003eAlpi\u003c/em\u003e, \u003cem\u003eFabp1\u003c/em\u003e, \u003cem\u003eSlc5a1\u003c/em\u003e, and \u003cem\u003eSlc2a2\u003c/em\u003e for enterocytes, \u003cem\u003eChga\u003c/em\u003e for EECs, \u003cem\u003eLyz1\u003c/em\u003e for Paneth cells and goblet cells, \u003cem\u003eMuc2 \u003c/em\u003efor goblet cells, and\u003cem\u003e Dclk\u003c/em\u003e1 for tuft cells.\u003c/p\u003e","description":"","filename":"251107AgingPaperFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8093838/v1/7ba96836d1269eaec7b28fb9.png"},{"id":97137461,"identity":"aba14c6b-3b1b-4dad-8fc9-25236f4b6c00","added_by":"auto","created_at":"2025-12-01 09:57:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":270374,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePseudotime analysis of intestinal epithelial organoids derived from young and aged mice: \u003c/strong\u003e(A) UMAP plots showing pseudotime trajectories of intestinal epithelial cells derived from organoids of young (left) and aged (right) mice. The color gradient represents pseudotime progression from stem cells toward differentiated lineages. The starting point of pseudotime is indicated by arrows. (B) Cell ratio distributions along normalized pseudotime for representative cell types—intestinal stem cells, TA cells, enterocytes, and EECs—in organoids derived from young and aged mice. In all examined cell types, mean pseudotime values were significantly higher in organoids derived from aged mice compared with those from young mice (p \u0026lt; 0.001), indicating age-associated delays in epithelial differentiation \u003cem\u003eex vivo\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"251107AgingPaperFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8093838/v1/00334cb3ca1dc947e0476cbf.png"},{"id":97139025,"identity":"1a09e2a1-a48e-41e2-8706-f1e2468e6a71","added_by":"auto","created_at":"2025-12-01 09:59:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":189379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003escRNA-seq analysis of tissue-derived intestinal epithelial cells\u003c/strong\u003e: (A) UMAP plot showing clustering of cells from scRNA-seq analysis of tissue-derived intestinal epithelial cells. “Total” indicates the combined dataset of young and aged samples. Cluster labels indicate cell types. TA cells, transit-amplifying cells; Stem-TA cells indicate cells within this cluster have both expression of stem-cell-marker genes and TA-cell-marker genes; AEPs, absorptive epithelial progenitor cells; SPCs, secretory epithelial progenitor cells; EECs, enteroendocrine cells. (B) The marker genes used for identifying each cell type are as follows: \u003cem\u003eLgr5\u003c/em\u003e and \u003cem\u003eOlfm4\u003c/em\u003e for stem cells (ISCs), \u003cem\u003eMki67\u003c/em\u003e for TA cells, Hes1 for \u003cem\u003eAEPs\u003c/em\u003e, \u003cem\u003eAlpi\u003c/em\u003e, \u003cem\u003eFabp1\u003c/em\u003e, \u003cem\u003eSlc5a1\u003c/em\u003e, and \u003cem\u003eSlc2a2\u003c/em\u003e for enterocytes, \u003cem\u003eChga\u003c/em\u003e for EECs, \u003cem\u003eLyz1\u003c/em\u003e for Paneth cells and goblet cells, \u003cem\u003eMuc2\u003c/em\u003e for goblet cells, and \u003cem\u003eDclk1\u003c/em\u003e for tuft cells.\u003c/p\u003e","description":"","filename":"251107AgingPaperFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8093838/v1/22ee943b353664b1b475426e.png"},{"id":96987557,"identity":"69b65ddf-83e6-4f21-b0a3-24cfa6b87185","added_by":"auto","created_at":"2025-11-28 10:33:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":331031,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePseudotime analysis of tissue-derived intestinal epithelial cells derived from young and aged mouse tissues:\u003c/strong\u003e (A) UMAP plots showing pseudotime trajectories of intestinal epithelial cells obtained from young (left) and aged (right) mice. The color gradient represents pseudotime progression from stem cells toward differentiated lineages. The starting point of pseudotime is indicated by arrows. (B) Cell ratio distributions along normalized pseudotime for representative cell types—intestinal stem cells, TA cells, and tuft cells—in young and aged mice. In all examined cell types, the mean pseudotime values were significantly higher in aged mice compared with young mice (p \u0026lt; 0.001), indicating delayed differentiation during intestinal epithelial aging.\u003c/p\u003e","description":"","filename":"251107AgingPaperFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8093838/v1/30a1614e3cb3721982c0482a.png"},{"id":96987543,"identity":"80621994-6336-4d2e-bb75-6adcc4fed956","added_by":"auto","created_at":"2025-11-28 10:33:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1299294,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of the number of spherical organoids in Matrigel:\u003c/strong\u003e (A) Representative images of intestinal epithelial organoids derived from young and aged mice at day 3 after subculture. Spherical organoids (defined as organoids without any budding structures) are indicated by red arrows. (B) Temporal changes in the number of spherical organoids relative to the total organoid area were quantified on days 3 to 6 after subculture. Organoids derived from aged mice exhibited a significantly higher proportion of spherical organoids compared with those from young mice (p = 0.0363), indicating delayed budding formation and impaired differentiation with aging.\u003c/p\u003e","description":"","filename":"251107AgingPaperFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8093838/v1/6fdd6ba260476994867cd901.png"},{"id":97138781,"identity":"c4e7d3fb-ce14-4ca3-8f8c-e2293d74ee43","added_by":"auto","created_at":"2025-12-01 09:59:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":65507,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTemporal changes in relative expression levels of marker genes in intestinal epithelial organoids derived from young and aged mice.: \u003c/strong\u003eRelative mRNA expression levels of \u003cem\u003eLgr5\u003c/em\u003e(ISC marker), \u003cem\u003eMki67\u003c/em\u003e (TA cell marker), \u003cem\u003eAlpi\u003c/em\u003e and \u003cem\u003eSlc2a2\u003c/em\u003e(enterocyte markers), \u003cem\u003eLyz1\u003c/em\u003e (Paneth cell marker), \u003cem\u003eMuc2\u003c/em\u003e (goblet cell marker), \u003cem\u003eDclk1\u003c/em\u003e (tuft cell marker), and \u003cem\u003eChga\u003c/em\u003e (EEC marker) were quantified by RT-qPCR on days 3 to 6 after passage. Expression levels were normalized to \u003cem\u003eGapdh\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"251109AgingPaperFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-8093838/v1/67bd03365a879a77cd45c7c8.png"},{"id":96987547,"identity":"82d52e9e-7d5f-41de-9089-aa5ba920bd2b","added_by":"auto","created_at":"2025-11-28 10:33:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":91571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation between temporal changes in marker gene expression and the number of spherical organoids: \u003c/strong\u003eSpearman’s rank correlation coefficients (ρ) were calculated to evaluate the relationship between relative expression levels of marker genes (\u003cem\u003eLgr5, Mki67, Alpi, Slc2a2, Lyz1, Muc2, Dclk1,\u003c/em\u003e and \u003cem\u003eChga\u003c/em\u003e) and the number of spherical organoids measured from days 3 to 6 after passage. The correlation coefficient (ρ) and corresponding p value are indicated in each panel. Coefficients from the linear mixed-effects model (Coef) represent the estimated change in the number of spherical organoids per unit change in gene expression.\u003c/p\u003e","description":"","filename":"251109AgingPaperFig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-8093838/v1/7b28b85ca89b60805649b7cf.png"},{"id":97248809,"identity":"842b7bb7-a9fd-410b-825d-cf50375f8c9c","added_by":"auto","created_at":"2025-12-02 13:07:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4115225,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8093838/v1/45724a8a-8da1-4591-973f-330a08110c49.pdf"},{"id":96987541,"identity":"3293d2a3-6068-4be0-8524-d03ba1e67f0d","added_by":"auto","created_at":"2025-11-28 10:33:49","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15782,"visible":true,"origin":"","legend":"","description":"","filename":"251109AgingPaperSupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8093838/v1/85a1919674f2f95f976c75b4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Single-cell analysis of intestinal epithelial organoids reveals aging-associated differentiation delay conserved between in vivo and ex vivo","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe intestinal epithelium is one of the most rapidly renewing tissues in the body, and this renewal is sustained by intestinal epithelial stem cells (ISCs) located at the base of the crypts (Rodriguez-Colman et al., 2017). These cells give rise to proliferative transit-amplifying (TA) cells, which further differentiate into various specialized epithelial lineages, including enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. The precise regulation of ISC self-renewal and differentiation is essential for maintaining intestinal homeostasis.\u003c/p\u003e\n\u003cp\u003eAging is associated with a progressive decline in the regenerative capacity of many tissues, including the intestine. Previous studies have suggested that aging leads to reduced stem cell function, altered lineage allocation, and increased susceptibility to inflammation and disease (Oh, Lee, \u0026amp; Wagers, 2014) . In the intestinal epithelium, aging can alter crypt architecture, reduce Lgr5+ ISC abundance, and impair Wnt signaling (Nalapareddy et al., 2017; Pentinmikko et al., 2019). Additionally, aged ISCs exhibit impaired responses to injury and diminished organoid-forming capacity (Igarashi et al., 2019). Transcriptional profiling studies have further revealed age-associated changes in stem cell identity and epigenetic regulation (Choi et al., 2023; Moor et al., 2017) .\u003c/p\u003e\n\u003cp\u003eDespite these insights, the specific cellular and molecular mechanisms through which aging impairs ISC differentiation remain unclear. In particular, the early transition from stem cells to TA cells is critical for epithelial turnover but has not been well-characterized in the context of aging.\u003c/p\u003e\n\u003cp\u003eTo address these questions, we performed single-cell RNA sequencing (scRNA-seq) to profile organoids derived from intestinal crypts of young and aged mice. Organoids serve as powerful \u003cem\u003eex vivo\u003c/em\u003e models that recapitulate key features of the intestinal epithelium, including cell-type diversity and stem cell hierarchies. We complemented our analysis with scRNA-seq data from native intestinal tissues to assess the physiological relevance of the observed organoid changes. Our findings revealed age-associated delays in ISC differentiation, particularly at early steps, and established a framework for using organoids to model epithelial aging.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eEstablishment of intestinal epithelial organoids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolation and dissociation of stem cells from the normal intestinal epithelium of wild-type C57BL/6 mice were performed as previously described (Uchida et al., 2019) . Isolated epithelial cells were embedded in Matrigel on ice (growth factor-reduced, phenol red-free; Corning, Corning, NY, USA) and seeded in 48-well plates. The Matrigel was polymerized for 10 min at 37 °C, and overlaid with 250 μL/well basal culture medium (advanced Dulbecco’s modified Eagle medium/F12 supplemented with penicillin/streptomycin, 10 mmol/L HEPES, Glutamax, 1 × N2, 1 × B27 [all from Thermo Fisher Scientific, Waltham, MA, USA], and 1 mmol/L N-acetylcysteine [Sigma-Aldrich, St. Louis, MO, USA]) containing the following optimized growth factor combinations: murine epidermal growth factor (Thermo Fisher Scientific), recombinant murine noggin (Peprotech, Rocky Hill, New Jersey, USA), Y-27632 (Selleck, Yokohama, Kanagawa, Japan), and R-spondin 1. In this study, mice older than 50 weeks were considered aged, and the establishment of intestinal epithelial organoids was deemed successful when cultures were maintained for over five passages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003escRNA-seq analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor scRNA-seq analysis, we used organoid-derived intestinal epithelial cells from 21-week-old (young) and 112-week-old (aged) mice. Organoids were dissociated into single cells using TrypLE (Thermo Fisher Scientific), filtered through a cell strainer, and resuspended in 2% BSA/PBS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003escRNA-seq was performed using the Chromium Next GEM Single Cell 3’ Kit v3.1 with Dual Index (10x Genomics, Pleasanton, CA, USA), following the manufacturer’s protocol. Briefly, single-cell suspensions were loaded onto the Chromium Controller to generate Gel Bead-in-Emulsions (GEMs), and reverse transcription was conducted within droplets. Following complementary DNA (cDNA) amplification, three-prime gene expression libraries with dual indexing were constructed. cDNA amplicons were enzymatically fragmented and size-selected to achieve optimal insert sizes. Subsequently, end repair, A-tailing, adaptor ligation, and PCR amplification were performed to incorporate the P5 and P7 flow cell adaptors, i7 and i5 sample indices, and TruSeq Read 2 primer sequence. The resulting libraries were sequenced on an Illumina NovaSeq 6000 platform with the following configuration: paired-end, dual indexing, Read 1: 28 cycles, Read 2: 90 cycles, Index read i7: 10 cycles, and Index read i5: 10 cycles, targeting a sequencing depth of 2,000 read pairs per cell. Base calls were converted to FASTQ files and aligned to the mouse reference genome using Cell Ranger to generate the gene-cell count matrix. Downstream data processing and analysis were performed using the Seurat R package on R. Low-quality cells, including those with \u0026lt;200 detected genes, \u0026gt;10% mitochondrial gene expression, or identified as potential doublets, were filtered out. After quality control, data were log-normalized and scaled to correct for technical variations, such as library size differences. For dimensionality reduction, principal component analysis (PCA) was performed using Seurat’s RunPCA function, and the number of significant principal components was selected based on the ElbowPlot. The data were further visualized using Uniform Manifold Approximation and Projection (UMAP). Cluster identification was conducted, and cell-type annotation was based on the expression of canonical marker genes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe also analyzed publicly available scRNA-seq datasets of intestinal epithelial tissues from young (5-month-old) and aged (24-month-old) mice, which are deposited in the Gene Expression Omnibus (GEO) database under accession number GSE210669 (Choi et al., 2023) .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePseudotime analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe utilized pseudotime analysis to reconstruct the differentiation trajectory of stem cells and to visualize gene expression changes associated with aging. Pseudotime analysis was performed using the Monocle 3 R package to infer the dynamic cellular trajectories during intestinal epithelial differentiation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll analyses were performed using R version 4.3.3, and statistical significance was set at p \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor passaged and cultured organoids, Cell Recovery Solution (Corning) was added to each well, and the plate was kept on ice for 1 h. Cells were centrifuged for 2 min, the supernatant was removed, and total RNA was extracted using QIAshredder (QIAGEN, Hilden, Germany) and the RNeasy Mini Kit (QIAGEN), according to the manufacturer’s instructions. The purified RNA was dissolved in 30 µL of RNase-free water and quantified using a NanoDrop 2000c spectrophotometer (Thermo Scientific).\u003c/p\u003e\n\u003cp\u003ecDNA was synthesized from the extracted RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) with RNase inhibitor, following the manufacturer's protocol. Reverse transcription was performed using a Mastercycler nexus gradient (Eppendorf, Hamburg, Germany).\u003c/p\u003e\n\u003cp\u003eRelative cDNA quantification was performed using the Pfaffl method. qPCR was carried out with PowerTrack™ SYBR Green Master Mix for qPCR (Thermo Fisher Scientific) in MicroAmp™ Optical 96-Well Reaction Plates (Thermo Fisher Scientific), and fluorescence was detected using the CFX Connect™ Real-Time PCR System (Bio-Rad laboratories, Hercules, CA, USA). Primer sequences are listed in Supplementary Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of temporal morphological changes of intestinal epithelial organoids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImages of organoids were acquired on days 3 to 6 after passaging. Image analysis was performed under blinded conditions. The number of spherical organoids (defined as organoids without any budding structures; organoids showing at least one bud were excluded) was visually counted in each image. This process was performed three times independently, and the images were subsequently unblinded to confirm their classification as young or old organoids. Finally, the mean number of spherical organoids per image was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were carried out using R. Comparisons between two groups were assessed using a two-tailed unpaired t-test. Welch's t-test was applied when variances were unequal and Student's t-test was used otherwise. For time-series data, differences between two groups were evaluated using a linear mixed-effects model with the Satterthwaite-adjusted F-test. Spearman's rank correlation was assessed using a t-distribution-based significance test. For all analyses, p values \u0026lt; 0.05 were considered statistically significant. For the graph in Figure 7, the regression line and its 95% confidence interval were generated using a Pearson correlation (linear regression), while the statistical significance of the association was assessed using Spearman's rank correlation to ensure robustness against non-linear relationships and potential outliers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was approved by the Animal Experiments and Experimental Animal Welfare Committee of Keio University (Approved No. A2022-105), and a proof/certificate of approval is available upon request.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEstablishment of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ei\u003c/strong\u003e\u003cstrong\u003entestinal\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003cstrong\u003epithelial\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003cstrong\u003erganoids and scRNA-seq\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003enalysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe established organoids using intestinal epithelial tissues obtained from mice at various ages. Representative images of a single stem cell expanding into organoids derived from intestinal epithelia of mice are shown in Figure 1A.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIntestinal epithelial organoids derived from young mice demonstrated vigorous growth and expanded through budding structures resembling crypts that harbor stem cells. In contrast, organoids derived from the intestinal epithelium of aged mice demonstrated a lower establishment efficiency. Moreover, once established, these organoids exhibited reduced proliferative capacity and limited budding formation (Figure 1A).\u003c/p\u003e\n\u003cp\u003eFigure 1B shows the UMAP results from single-cell analysis of intestinal epithelial organoids derived from both young and aged mice. The analysis revealed that the organoids were classified into ten distinct clusters, confirming that they contained all major intestinal epithelial cell types, including stem cells (ISCs), TA cells, absorptive enterocyte precursors (AEPs), secretory progenitor cells (SPCs), enterocytes, enteroendocrine cells (EECs), Paneth cells, goblet cells, and tuft cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 1C shows violin plots of the expression levels of marker genes for each cluster in the intestinal epithelial organoids. The marker genes used for identifying each cell type were as follows: \u003cem\u003eLgr5\u003c/em\u003e and \u003cem\u003eOlfm4\u003c/em\u003e for stem cells (ISCs), \u003cem\u003eMki67\u003c/em\u003e for TA cells, \u003cem\u003eHes1\u003c/em\u003e for AEPs, \u003cem\u003eAlpi,\u003c/em\u003e \u003cem\u003eFabp1\u003c/em\u003e, \u003cem\u003eSlc5a1\u003c/em\u003e, and \u003cem\u003eSlc2a2\u0026nbsp;\u003c/em\u003efor enterocytes, \u003cem\u003eChga\u003c/em\u003e for EECs, \u003cem\u003eLyz1\u003c/em\u003e for Paneth cells and goblet cells, \u003cem\u003eMuc2\u003c/em\u003e for goblet cells, and \u003cem\u003eDclk1\u003c/em\u003e for tuft cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePseudotime analysis using intestinal epithelial organoids derived from young and aged mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we performed pseudotime analysis using intestinal epithelial organoids derived from young and aged mice. Pseudotime analysis is a computational method used in scRNA-seq studies to infer the temporal order of cells along a biological process, such as differentiation or development. It does not measure actual time but instead estimates a trajectory that reflects how cells transition from one state to another based on their gene expression profiles.\u003c/p\u003e\n\u003cp\u003eFigure 2A shows the results of pseudotime analysis of intestinal epithelial organoids derived from young and aged mice. The arrows indicate the starting point of differentiation from stem cells. As shown in Figure 2A, organoids derived from young mice contain fewer early-stage stem cells but a higher proportion of cells at later stages of differentiation compared to those from aged mice. In contrast, organoids derived from aged mice have a larger number of early-stage stem cells and fewer cells at later stages of differentiation.\u003c/p\u003e\n\u003cp\u003eTherefore, we analyzed the relationship between pseudotime differentiation and the proportion of each cell type in intestinal epithelial organoids derived from young and aged mice. As shown in Figure 2B, all examined cell types—stem cells, TA cells, enterocytes, and EECs—in intestinal epithelial organoids derived from aged mice exhibited delayed differentiation compared to those from young mice.\u003c/p\u003e\n\u003cp\u003eThe pseudotime analysis revealed significant differences in differentiation trajectories between young and aged mice across all examined cell types:\u003c/p\u003e\n\u003cp\u003eStem cells: Mean pseudotime was 0.0875 in young and 0.2232 in aged mice (p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eTransit-amplifying (TA) cells: Mean pseudotime was 0.2285 in young and 0.5103 in aged mice (p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eEnterocytes: Mean pseudotime was 0.3734 in young and 0.6901 in aged mice (p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eEnteroendocrine cells (EECs): Mean pseudotime was 0.6341 in young and 0.9657 in aged mice (p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eThese results indicate a consistent delay in cellular differentiation in the aged intestinal epithelium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003escRNA-seq and pseudotime analyses using cells derived from intestinal epithelial tissues of young and aged mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we performed a similar analysis using publicly available scRNA-seq data of intestinal epithelial cells from young and aged mice published by Choi et al. (Choi et al., 2023) and compared the results with those obtained from our intestinal epithelial organoids.\u003c/p\u003e\n\u003cp\u003eFigure 3A shows the UMAP results from single-cell analysis of cells derived from intestinal epithelial tissues of young and aged mice. Similar to our organoid data, the single-cell analysis of tissue-derived cells revealed ten distinct clusters, confirming the presence of all major intestinal epithelial cell types, including stem cells (ISCs), TA cells, AEPs, SPCs, enterocytes, EECs, Paneth cells, goblet cells, and tuft cells.\u003c/p\u003e\n\u003cp\u003eFigure 3B shows violin plots of the expression levels of marker genes for each cluster in cells derived from intestinal epithelial tissue. The marker genes used to identify each cell type were as follows: \u003cem\u003eLgr5\u003c/em\u003e and \u003cem\u003eOlfm4\u003c/em\u003e for stem cells (ISCs), \u003cem\u003eMki67\u003c/em\u003e for TA cells, \u003cem\u003eHes1\u003c/em\u003e for AEPs, \u003cem\u003eAlpi,\u003c/em\u003e \u003cem\u003eFabp1\u003c/em\u003e, \u003cem\u003eSlc5a1\u003c/em\u003e, and \u003cem\u003eSlc2a2\u003c/em\u003e for enterocytes, \u003cem\u003eChga\u003c/em\u003e for EECs, \u003cem\u003eLyz1\u003c/em\u003e for Paneth cells and goblet cells, \u003cem\u003eMuc2\u003c/em\u003e for goblet cells, and \u003cem\u003eDclk1\u003c/em\u003e for tuft cells.\u003c/p\u003e\n\u003cp\u003eFigure 4A shows the results of pseudotime analysis using cells derived from the intestinal epithelial tissue of young and aged mice. The arrow indicates the starting point of differentiation from stem cells. In the tissue derived from young mice, a greater number of cells were observed at later stages of differentiation. In contrast, the tissue from aged mice exhibited fewer cells at the later stages, suggesting impaired or delayed differentiation with aging.\u003c/p\u003e\n\u003cp\u003eTo further assess the effects of aging on cellular differentiation in intestinal epithelial tissue, we conducted pseudotime analysis using cells derived from young and aged mice. As shown in Figure 4B, all examined cell types—stem cells, transit-amplifying (TA) cells, and tuft cells—exhibited significantly higher pseudotime values in aged mice compared to young mice, indicating delayed differentiation with aging:\u003c/p\u003e\n\u003cp\u003eStem cells, the mean pseudotime was 0.1059 in young mice and 0.1766 in aged mice (p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eTA cells, the mean pseudotime was 0.1445 in young mice and 0.4983 in aged mice (p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eTuft cells, the mean pseudotime was 0.7164 in young mice and 0.7714 in aged mice (p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eThese findings further supported the observation that aging is associated with a consistent delay in intestinal epithelial cell differentiation in both tissues and organoid models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTemporal morphological and marker gene expression changes in intestinal epithelial organoids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDelayed differentiation also is expected to affect budding formation, a characteristic feature of intestinal epithelial organoids. Therefore, to more definitively confirm the possibility of delayed differentiation suggested by the scRNA-seq results, we examined how it manifests in organoids \u003cem\u003eex vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIntestinal epithelial organoids are spherical immediately after passage, but gradually acquire the budding structures characteristic of intestinal epithelial organoids. After passage, we evaluated changes in organoid morphology by observing the growth of intestinal epithelial organoids over time and counting the number of spherical organoids relative to the total organoid area within the field of view. Figure 5A shows the counting process of spherical organoids and Figure 5B shows its temporal morphological changes. Figure 5B indicates that organoids with budding structures appeared earlier in young mice than in aged mice, while spherical organoids persisted longer in aged mice (p=0.0363). Budding structures are known to form with the appearance of Paneth cells, one of the final differentiated cell types (Sato et al., 2009; Sato et al. 2011; Serra et al., 2019), indicating that budding formation commences upon complete differentiation. Thus, delayed budding formation indicates delayed differentiation progression.\u003c/p\u003e\n\u003cp\u003eTo investigate changes in the expression of marker genes associated with morphological changes in intestinal epithelial organoids, we performed RNA extraction from organoids at days 3 to 6 after passage. RT-qPCR was performed for the marker genes and correlations between their expression levels and organoid morphology were calculated (Figure 6, Figure 7). As shown in Figure 6, expression of \u003cem\u003eMki67\u003c/em\u003e, a marker gene of TA cells, \u003cem\u003eMuc2\u003c/em\u003e, a marker gene of goblet cells, and \u003cem\u003eChga\u003c/em\u003e, a marker gene of EECs, was significantly higher in aged mice compared to young mice. Conversely, expression of \u003cem\u003eLyz1\u003c/em\u003e, which is a marker gene of Paneth cells, was significantly higher in young mice compared to aged mice. Furthermore, correlations between the expressions of the marker genes and the morphological changes in intestinal epithelial organoids were particularly strong for \u003cem\u003eLgr5\u003c/em\u003e, \u003cem\u003eSlc2a2\u003c/em\u003e, \u003cem\u003eLyz1\u003c/em\u003e, and \u003cem\u003eDclk1\u003c/em\u003e (rho≧0.7 or rho≦-0.7).\u0026nbsp;These results were consistent with previous research asserting that Paneth cell activity is necessary for budding formation in intestinal epithelial organoids (Sato et al., 2009; Sato et al., 2011; Serra et al., 2019).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provided comprehensive single-cell resolution insights into the effects of aging on intestinal epithelial stem cell differentiation. We demonstrated that aging impaired the early transition of ISCs to TA cells, leading to altered epithelial renewal. Importantly, these findings were consistent across both organoid models and native tissues, supporting the robustness and physiological relevance of the observations. Additionally, intestinal epithelial organoids exhibit an age-associated decline in differentiation capacity and function, manifested as delayed formation of budding structures.\u003c/p\u003e\n\u003cp\u003eThe results from pseudotime analysis of scRNA-seq data from intestinal epithelial tissue and intestinal epithelial organoids suggested that differentiation from ISCs to TA cells and later stages may be delayed with aging, both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e. Furthermore, although the intestinal epithelial tissue was derived from a hybrid mouse strain, similar results were obtained with intestinal epithelial organoids derived from C57BL/6J mice. This suggested that the observed aging changes occur to some extent regardless of genetic diversity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLyz1\u003c/em\u003e expression, a Paneth cell marker, showed a strong inverse correlation with the number of spherical organoids, and its increased expression was significantly associated with a decrease in the number of spherical organoids. This indicated that the activity level of Paneth cells, specifically those expressing \u003cem\u003eLyz1\u003c/em\u003e (where activity level refers to either higher cell counts or stronger cellular function), is strongly associated with budding formation in intestinal epithelial organoids. This aligned with previous research asserting that Paneth cell activity is necessary for budding formation in intestinal epithelial organoids (Sato et al., 2011; Sato et al., 2009; Serra et al., 2019) .\u003c/p\u003e\n\u003cp\u003eThe number of spherical organoids and RT-qPCR results suggested that age-related decline in Paneth cell function may delay differentiation from stem cells to TA cells and later stages. While Paneth cells exist in crypts alongside stem cells and are known to regulate stem cell differentiation by modulating Wnt and Notch signaling intensity, it is also known that while their number increases with aging, their function declines (Wallaeys, Garcia-Gonzalez, \u0026amp; Libert, 2023). Considering that pseudotime analysis in scRNA-seq assigns pseudotime to each cell in mature intestinal epithelial tissue and intestinal epithelial organoids, it is necessary to consider that dysfunctional Paneth cells may already exist around stem cells in these tissues and organoids, potentially affecting the stem cell transcriptome. Integrating our results with previous studies suggests that age-related changes in cell-cell interactions, particularly a decline in Paneth cell function, contribute to delayed differentiation from stem cells to TA cells and later stages.\u003c/p\u003e\n\u003cp\u003eOur pseudotime analysis with both organoid and \u003cem\u003ein vivo\u003c/em\u003e tissue data complements and extends prior research, offering a dynamic and comparative view of age-related stem cell dysfunction. By excluding systemic influences, organoids serve as ideal models to dissect epithelial-intrinsic aging mechanisms and to screen rejuvenation strategies. Intestinal epithelial organoids express the decline in differentiation potential and function into Paneth cells, which also occurs in intestinal epithelial tissue with aging, with the accompanying reduction in stem cell differentiation potential manifested as delayed formation of budding structures. In other words, the differentiation capacity of intestinal epithelial organoids reflects age-related changes in intestinal epithelial tissue differentiation. By focusing on delayed budding structure formation and differentiation of cell marker genes, it may be possible to conduct aging research \u003cem\u003eex vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn the broader context of aging biology, our findings underscore the critical role of stem cell dynamics in tissue homeostasis and age-related pathologies. Understanding how aging disrupts ISC behavior may inform strategies to enhance intestinal regeneration and resilience in the elderly.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIntestinal epithelial organoids faithfully modeled age-related impairments in stem cell differentiation. Aging delayed the transition of ISCs to TA cells and later stages, reflecting a key vulnerability in epithelial renewal. Our study established a foundation for future research into intestinal aging and provided a tractable system for therapeutic development. Moreover, our study suggested that organoids have potential as models of stem cell-derived tissue aging.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Animal Facility staff for their daily care of the laboratory animals. Also, we would like to thank Editage (www.editage.jp) for English language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI (Grant Numbers JP24KK0207 and JP24K03285) grants awarded to Y.S.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.H., Masaya K., Masaki K., J.M., J.N., and Y.S. designed the experiments and supervised the work. K.T., R.Y., and T.M. performed data processing. K.H. and Masaya K. performed the experiments and statistical analysis. K.H., Masaya K., and Y.S. generated the figures. \u0026nbsp;K.H., T.I., and Y.S. wrote and edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors declare that there are no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eChoi, J., Houston, M., Wang, R., Ye, K., Li, W., Zhang, X., . . . Augenlicht, L. H. (2023). Intestinal stem cell aging at single-cell resolution: Transcriptional perturbations alter cell developmental trajectory reversed by gerotherapeutics. \u003cem\u003eAging Cell, 22\u003c/em\u003e(5), e13802. doi:10.1111/acel.13802\u003c/li\u003e\n \u003cli\u003eIgarashi, M., Miura, M., Williams, E., Jaksch, F., Kadowaki, T., Yamauchi, T., \u0026amp; Guarente, L. (2019). NAD(+) supplementation rejuvenates aged gut adult stem cells. \u003cem\u003eAging Cell, 18\u003c/em\u003e(3), e12935. doi:10.1111/acel.12935\u003c/li\u003e\n \u003cli\u003eMoor, A. E., Golan, M., Massasa, E. E., Lemze, D., Weizman, T., Shenhav, R., . . . Itzkovitz, S. (2017). Global mRNA polarization regulates translation efficiency in the intestinal epithelium. \u003cem\u003eScience, 357\u003c/em\u003e, 1299-1303. doi:10.1126/science.aan2399\u003c/li\u003e\n \u003cli\u003eNalapareddy, K., Nattamai, K. J., Kumar, R. S., Karns, R., Wikenheiser-Brokamp, K. A., Sampson, L. L., . . . Geiger, H. (2017). Canonical Wnt Signaling Ameliorates Aging of Intestinal Stem Cells. \u003cem\u003eCell Reports, 18\u003c/em\u003e(11), 2608-2621. doi:10.1016/j.celrep.2017.02.056\u003c/li\u003e\n \u003cli\u003eOh, J., Lee, Y. D., \u0026amp; Wagers, A. J. (2014). Stem cell aging: mechanisms, regulators and therapeutic opportunities. \u003cem\u003eNature Medicine, 20\u003c/em\u003e(8), 870-880. doi:10.1038/nm.3651\u003c/li\u003e\n \u003cli\u003ePentinmikko, N., Iqbal, S., Mana, M., Andersson, S., Cognetta, A. B., 3rd, Suciu, R. M., . . . Katajisto, P. (2019). Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium. \u003cem\u003eNature, 571\u003c/em\u003e(7765), 398-402. doi:10.1038/s41586-019-1383-0\u003c/li\u003e\n \u003cli\u003eRodriguez-Colman, M. J., Schewe, M., Meerlo, M., Stigter, E., Gerrits, J., Pras-Raves, M., . . . Burgering, B. M. (2017). Interplay between metabolic identities in the intestinal crypt supports stem cell function. \u003cem\u003eNature, 543\u003c/em\u003e(7645), 424-427. doi:10.1038/nature21673\u003c/li\u003e\n \u003cli\u003eSato, T., van Es, J. H., Snippert, H. J., Stange, D. E., Vries, R. G., van den Born, M., . . . Clevers, H. (2011). Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. \u003cem\u003eNature, 469\u003c/em\u003e(7330), 415-418. doi:10.1038/nature09637\u003c/li\u003e\n \u003cli\u003eSato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., . . . Clevers, H. (2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. \u003cem\u003eNature, 459\u003c/em\u003e(7244), 262-265. doi:10.1038/nature07935\u003c/li\u003e\n \u003cli\u003eSerra, D., Mayr, U., Boni, A., Lukonin, I., Rempfler, M., Challet Meylan, L., . . . Liberali, P. (2019). Self-organization and symmetry breaking in intestinal organoid development. \u003cem\u003eNature, 569\u003c/em\u003e(7754), 66-72. doi:10.1038/s41586-019-1146-y\u003c/li\u003e\n \u003cli\u003eUchida, R., Saito, Y., Nogami, K., Kajiyama, Y., Suzuki, Y., Kawase, Y., . . . Saito, H. (2019). Erratum: Publisher Correction: Epigenetic silencing of Lgr5 induces senescence of intestinal epithelial organoids during the process of aging. \u003cem\u003eNPJ Aging Mech Dis, 5\u003c/em\u003e, 5. doi:10.1038/s41514-019-0035-9\u003c/li\u003e\n \u003cli\u003eWallaeys, C., Garcia-Gonzalez, N., \u0026amp; Libert, C. (2023). Paneth cells as the cornerstones of intestinal and organismal health: a primer. \u003cem\u003eEMBO Molecular Medicine, 15\u003c/em\u003e(2), e16427. doi:10.15252/emmm.202216427\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-aging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Aging](https://www.nature.com/npjamd/)","snPcode":"41514","submissionUrl":"https://submission.springernature.com/new-submission/41514/3","title":"npj Aging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8093838/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8093838/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Intestinal epithelial stem cells (ISCs) are essential for maintaining the structural and functional integrity of the gut by continuously replenishing the intestinal epithelium. However, the effects of aging on the identity and function of ISCs and their progeny remain poorly understood. In this study, we performed single-cell RNA sequencing (scRNA-seq) to analyze the cellular composition and transcriptional landscape of intestinal epithelial organoids derived from young and aged mice. To complement and validate our organoid findings, we also examined publicly available scRNA-seq datasets from intestinal epithelial tissues of mice across different age groups. Our analysis revealed that ISCs from aged mice exhibited impaired differentiation trajectories, with a pronounced delay in the transition to transit-amplifying (TA) cells and later stages. Remarkably, the same differentiation delay and transcriptional changes were observed in native intestinal tissues, indicating that organoids can serve as faithful models for studying aging of stem cell-derived epithelial tissue. Additionally, a delay in differentiation was also observed in organoids derived from aged mice, manifested as a delayed onset of budding structures. Our results presented fundamental insights into the aging of ISCs conserved between in vivo and ex vivo and established an ex vivo platform for evaluating therapeutic interventions targeting intestinal regeneration in aging.","manuscriptTitle":"Single-cell analysis of intestinal epithelial organoids reveals aging-associated differentiation delay conserved between in vivo and ex vivo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-28 10:33:44","doi":"10.21203/rs.3.rs-8093838/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-04T16:11:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-04T14:16:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-03T20:20:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-29T07:30:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136883153083870792642482173965716242331","date":"2025-11-27T00:05:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114067923895596227764259187606181256276","date":"2025-11-25T20:53:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247805785289600849956955329599903256651","date":"2025-11-24T13:55:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-24T13:05:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-19T16:09:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-17T05:48:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Aging","date":"2025-11-12T08:18:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-aging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Aging](https://www.nature.com/npjamd/)","snPcode":"41514","submissionUrl":"https://submission.springernature.com/new-submission/41514/3","title":"npj Aging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8ce89643-0e87-43ab-b27f-8e115f231556","owner":[],"postedDate":"November 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":58678342,"name":"Biological sciences/Cell biology"},{"id":58678343,"name":"Biological sciences/Developmental biology"},{"id":58678344,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2025-12-04T16:23:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-28 10:33:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8093838","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8093838","identity":"rs-8093838","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}