Time-resolved fate mapping identifies the intestinal upper crypt zone as an origin of Lgr5+ crypt base columnar cells.

In the prevailing model, Lgr5+ cells are the only intestinal stem cells (ISCs) that sustain homeostatic epithelial regeneration by upward migration of progeny through elusive upper crypt transit-amplifying (TA) intermediates. Here, we identify a proliferative upper crypt population marked by Fgfbp1, in the location of putative TA cells, that is transcriptionally distinct from Lgr5+ cells. Using a kinetic reporter for time-resolved fate mapping and Fgfbp1-CreERT2 lineage tracing, we establish that Fgfbp1+ cells are multi-potent and give rise to Lgr5+ cells, consistent with their ISC function. Fgfbp1+ cells also sustain epithelial regeneration following Lgr5+ cell depletion. We demonstrate that FGFBP1, produced by the upper crypt cells, is an essential factor for crypt proliferation and epithelial homeostasis. Our findings support a model in which tissue regeneration originates from upper crypt Fgfbp1+ cells that generate progeny propagating bi-directionally along the crypt-villus axis and serve as a source of Lgr5+ cells in the crypt base.

LGR5 expressing skin fibroblasts define a major cellular hub perturbed in scleroderma.

Systemic sclerosis (scleroderma, SSc) is an incurable autoimmune disease with high morbidity and mortality rates. Here, we conducted a population-scale single-cell genomic analysis of skin and blood samples of 56 healthy controls and 97 SSc patients at different stages of the disease. We found immune compartment dysfunction only in a specific subtype of diffuse SSc patients but global dysregulation of the stromal compartment, particularly in a previously undefined subset of LGR5+-scleroderma-associated fibroblasts (ScAFs). ScAFs are perturbed morphologically and molecularly in SSc patients. Single-cell multiome profiling of stromal cells revealed ScAF-specific markers, pathways, regulatory elements, and transcription factors underlining disease development. Systematic analysis of these molecular features with clinical metadata associates specific ScAF targets with disease pathogenesis and SSc clinical traits. Our high-resolution atlas of the sclerodermatous skin spectrum will enable a paradigm shift in the understanding of SSc disease and facilitate the development of biomarkers and therapeutic strategies.

Snake Venom Gland Organoids.

Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.

Long-Term Culture Captures Injury-Repair Cycles of Colonic Stem Cells.

The colonic epithelium can undergo multiple rounds of damage and repair, often in response to excessive inflammation. The responsive stem cell that mediates this process is unclear, in part because of a lack of in vitro models that recapitulate key epithelial changes that occur in vivo during damage and repair. Here, we identify a Hopx+ colitis-associated regenerative stem cell (CARSC) population that functionally contributes to mucosal repair in mouse models of colitis. Hopx+ CARSCs, enriched for fetal-like markers, transiently arose from hypertrophic crypts known to facilitate regeneration. Importantly, we established a long-term, self-organizing two-dimensional (2D) epithelial monolayer system to model the regenerative properties and responses of Hopx+ CARSCs. This system can reenact the "homeostasis-injury-regeneration" cycles of epithelial alterations that occur in vivo. Using this system, we found that hypoxia and endoplasmic reticulum stress, insults commonly present in inflammatory bowel diseases, mediated the cyclic switch of cellular status in this process.

FXR Regulates Intestinal Cancer Stem Cell Proliferation.

Increased levels of intestinal bile acids (BAs) are a risk factor for colorectal cancer (CRC). Here, we show that the convergence of dietary factors (high-fat diet) and dysregulated WNT signaling (APC mutation) alters BA profiles to drive malignant transformations in Lgr5-expressing (Lgr5+) cancer stem cells and promote an adenoma-to-adenocarcinoma progression. Mechanistically, we show that BAs that antagonize intestinal farnesoid X receptor (FXR) function, including tauro-ß-muricholic acid (T-ßMCA) and deoxycholic acid (DCA), induce proliferation and DNA damage in Lgr5+ cells. Conversely, selective activation of intestinal FXR can restrict abnormal Lgr5+ cell growth and curtail CRC progression. This unexpected role for FXR in coordinating intestinal self-renewal with BA levels implicates FXR as a potential therapeutic target for CRC.

IL-17RA-signaling in Lgr5+ intestinal stem cells induces expression of transcription factor ATOH1 to promote secretory cell lineage commitment.

The Th17 cell-lineage-defining cytokine IL-17A contributes to host defense and inflammatory disease by coordinating multicellular immune responses. The IL-17 receptor (IL-17RA) is expressed by diverse intestinal cell types, and therapies targeting IL-17A induce adverse intestinal events, suggesting additional tissue-specific functions. Here, we used multiple conditional deletion models to identify a role for IL-17A in secretory epithelial cell differentiation in the gut. Paneth, tuft, goblet, and enteroendocrine cell numbers were dependent on IL-17A-mediated induction of the transcription factor ATOH1 in Lgr5+ intestinal epithelial stem cells. Although dispensable at steady state, IL-17RA signaling in ATOH1+ cells was required to regenerate secretory cells following injury. Finally, IL-17A stimulation of human-derived intestinal organoids that were locked into a cystic immature state induced ATOH1 expression and rescued secretory cell differentiation. Our data suggest that the cross talk between immune cells and stem cells regulates secretory cell lineage commitment and the integrity of the mucosa.

Gastric stem cells promote inflammation and gland remodeling in response to Helicobacter pylori via Rspo3-Lgr4 axis.

Helicobacter pylori is a pathogen that colonizes the stomach and causes chronic gastritis. Helicobacter pylori can colonize deep inside gastric glands, triggering increased R-spondin 3 (Rspo3) signaling. This causes an expansion of the "gland base module," which consists of self-renewing stem cells and antimicrobial secretory cells and results in gland hyperplasia. The contribution of Rspo3 receptors Lgr4 and Lgr5 is not well explored. Here, we identified that Lgr4 regulates Lgr5 expression and is required for H. pylori-induced hyperplasia and inflammation, while Lgr5 alone is not. Using conditional knockout mice, we reveal that R-spondin signaling via Lgr4 drives proliferation of stem cells and also induces NF-κB activity in the proliferative stem cells. Upon exposure to H. pylori, the Lgr4-driven NF-κB activation is responsible for the expansion of the gland base module and simultaneously enables chemokine expression in stem cells, resulting in gland hyperplasia and neutrophil recruitment. This demonstrates a connection between R-spondin-Lgr and NF-κB signaling that links epithelial stem cell behavior and inflammatory responses to gland-invading H. pylori.

WNT Oncogenic Transcription Requires MYC Suppression of Lysosomal Activity and EPCAM Stabilization in Gastric Tumors.

BACKGROUND & AIMS: WNT signaling is central to spatial tissue arrangement and regulating stem cell activity, and it represents the hallmark of gastrointestinal cancers. Although its role in driving intestinal tumors is well characterized, WNT's role in gastric tumorigenesis remains elusive. METHODS: We have developed mouse models to control the specific expression of an oncogenic form of ß-catenin (CTNNB1) in combination with MYC activation in Lgr5+ cells of the gastric antrum. We used multiomics approaches applied in vivo and in organoid models to characterize their cooperation in driving gastric tumorigenesis. RESULTS: We report that constitutive ß-catenin stabilization in the stomach has negligible oncogenic effects and requires MYC activation to induce gastric tumor formation. Although physiologically low MYC levels in gastric glands limit ß-catenin transcriptional activity, increased MYC expression unleashes the WNT oncogenic transcriptional program, promoting ß-catenin enhancer invasion without a direct transcriptional cooperation. MYC activation induces a metabolic rewiring that suppresses lysosomal biogenesis through mTOR and ERK activation and MiT/TFE inhibition. This prevents EPCAM degradation by macropinocytosis, promoting ß-catenin chromatin accumulation and activation of WNT oncogenic transcription. CONCLUSION: Our results uncovered a new signaling framework with important implications for the control of gastric epithelial architecture and WNT-dependent oncogenic transformation.

Direct Reprogramming of Hepatocytes Into JAK/Stat-Dependent LGR5+ Liver Cells Able to Initiate Intrahepatic Cholangiocarcinoma.

Somatic cells that have been partially reprogrammed by the factors Oct4, Sox2, Klf4, and cMyc (OSKM) have been demonstrated to be potentially tumorigenic in vitro and in vivo due to the acquisition of cancer-associated genomic alterations and the absence of OSKM clearance over time. In the present study, we obtained partially reprogrammed, SSEA1-negative cells by transducing murine hepatocytes with Δ1Δ3-deleted adenoviruses that expressed the 4 OSKM factors. We observed that, under long-term 2D and 3D culture conditions, hepatocytes could be converted into LGR5-positive cells with self-renewal capacity that was dependent on 3 cross-signaling pathways: IL6/Jak/Stat3, LGR5/R-spondin, and Wnt/ß-catenin. Following engraftment in syngeneic mice, LGR5-positive cells that expressed the cancer markers CD51, CD166, and CD73 were capable of forming invasive and metastatic tumors reminiscent of intrahepatic cholangiocarcinoma (ICC): they were positive for CK19 and CK7, featured associations of cord-like structures, and contained cuboidal and atypical cells with dissimilar degrees of pleomorphism and mitosis. The LGR5+-derived tumors exhibited a highly vascularized stroma with substantial fibrosis. In addition, we identified pro-angiogenic factors and signaling pathways involved in neo-angiogenesis and vascular development, which represent potential new targets for anti-angiogenic strategies to overcome tumor resistance to current ICC treatments.

NEDD4 and NEDD4L regulate Wnt signalling and intestinal stem cell priming by degrading LGR5 receptor.

The intestinal stem cell (ISC) marker LGR5 is a receptor for R-spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self-renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis, regeneration and cancer, little is known about the post-translational regulation of LGR5. Here, we show that the HECT-domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation, increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high-grade adenomas. Mechanistically, we find that both NEDD4 and NEDD4L negatively regulate Wnt/ß-catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post-translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers, which subsequently enhances tumour predisposition and progression.

A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease.

Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence-activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5-H2B-GFP and wild-type pigs. Ileum and colon LGR5-H2B-GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5-H2B-GFP colonoids. Crypt-base, green fluorescent protein (GFP) expressing cells co-localized with ISC biomarkers. LGR5-H2B-GFPhi cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5-H2B-GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5-H2B-GFP pig crypt-base cells. LGR5-H2B-GFP/APCnull colonoids had cystic growth in WNT/R-spondin-depleted media and significantly upregulated WNT/ß-catenin target gene expression (p < .05). LGR5+ ISCs are reproducibly isolated in LGR5-H2B-GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt-base FISH, underscore the significance of this novel LGR5-H2B-GFP pig to translational ISC research.

CDK4/6 inhibitor modulating active and quiescent intestinal stem cells for prevention of chemotherapy-induced diarrhea.

Chemotherapy-induced diarrhea causes dehydration, debilitation, infection, and even death, but there are currently no Food and Drug Administration (FDA)-approved drugs for treatment of chemotherapy-induced diarrhea. It is generally believed that the timely regulation of intestinal stem cell (ISC) fate may provide a meaningful solution for intestinal injuries. However, the lineage plasticity of ISCs during and after chemotherapy remains poorly understood. Here, we demonstrated that palbociclib, a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, regulated the fate of active or quiescent ISCs, provided multilineage protection from the toxicity of several different chemotherapeutics, and accelerated gastrointestinal epithelium recovery. Consistent with in vivo results, we determined that palbociclib enhanced intestinal organoid and ex vivo tissue survival after chemotherapy. Lineage tracing studies have shown that palbociclib protects active ISCs marked by Lgr5 and Olfm4 during chemotherapy and unexpectedly activates quiescent ISCs marked by Bmi1 to immediately participate in crypt regeneration after chemotherapy. Furthermore, palbociclib does not decrease the efficacy of cytotoxic chemotherapy in tumor grafts. The experimental evidence suggests that the combination of CDK4/6 inhibitors with chemotherapy could reduce damage to the gastrointestinal epithelium in patients. © 2023 The Pathological Society of Great Britain and Ireland.

Azoxymethane-induced carcinogenesis-like model of mouse intestine and mouse embryonic stem cell-derived intestinal organoids.

BACKGROUND: Tumor modeling using organoids holds potential in studies of cancer development, enlightening both the intracellular and extracellular molecular mechanisms behind different cancer types, biobanking, and drug screening. Intestinal organoids can be generated in vitro using a unique type of adult stem cells which are found at the base of crypts and are characterized by their high Lgr5 expression levels. METHODS AND RESULTS: In this study, we successfully established intestinal cancer organoid models by using both the BALB/c derived and mouse embryonic stem cells (mESCs)-derived intestinal organoids. In both cases, carcinogenesis-like model was developed by using azoxymethane (AOM) treatment. Carcinogenesis-like model was verified by H&E staining, immunostaining, relative mRNA expression analysis, and LC/MS analysis. The morphologic analysis demonstrated that the number of generated organoids, the number of crypts, and the intensity of the organoids were significantly augmented in AOM-treated intestinal organoids compared to non-AOM-treated ones. Relative mRNA expression data revealed that there was a significant increase in both Wnt signaling pathway-related genes and pluripotency transcription factors in the AOM-induced intestinal organoids. CONCLUSION: We successfully developed simple carcinogenesis-like models using mESC-based and Lgr5 + stem cell-based intestinal organoids. Intestinal organoid based carcinogenesi models might be used for personalized cancer therapy in the future.

RTCB deficiency triggers colitis in mice by influencing the NF-κB and Wnt/ß-catenin signaling pathways.

RNA terminal phosphorylase B (RTCB) has been shown to play a significant role in multiple physiological processes. However, the specific role of RTCB in the mouse colon remains unclear. In this study, we employ a conditional knockout mouse model to investigate the effects of RTCB depletion on the colon and the potential molecular mechanisms. We assess the efficiency and phenotype of Rtcb knockout using PCR, western blot analysis, histological staining, and immunohistochemistry. Compared with the control mice, the Rtcb-knockout mice exhibit compromised colonic barrier integrity and prominent inflammatory cell infiltration. In the colonic tissues of Rtcb-knockout mice, the protein levels of TNF-α, IL-8, and p-p65 are increased, whereas the levels of IKKß and IκBα are decreased. Moreover, the level of GSK3ß is increased, whereas the levels of Wnt3a, ß-catenin, and LGR5 are decreased. Collectively, our findings unveil a close association between RTCB and colonic tissue homeostasis and demonstrate that RTCB deficiency can lead to dysregulation of both the NF-κB and Wnt/ß-catenin signaling pathways in colonic cells.

A genome-scale CRISPR screen reveals factors regulating Wnt-dependent renewal of mouse gastric epithelial cells.

An ability to safely harness the powerful regenerative potential of adult stem cells for clinical applications is critically dependent on a comprehensive understanding of the underlying mechanisms regulating their activity. Epithelial organoid cultures accurately recapitulate many features of in vivo stem cell-driven epithelial renewal, providing an excellent ex vivo platform for interrogation of key regulatory mechanisms. Here, we employed a genome-scale clustered, regularly interspaced, short palindromic repeats (CRISPR) knockout (KO) screening assay using mouse gastric epithelial organoids to identify modulators of Wnt-driven stem cell-dependent epithelial renewal in the gastric mucosa. In addition to known Wnt pathway regulators, such as Apc, we found that KO of Alk, Bclaf3, or Prkra supports the Wnt independent self-renewal of gastric epithelial cells ex vivo. In adult mice, expression of these factors is predominantly restricted to non-Lgr5-expressing stem cell zones above the gland base, implicating a critical role for these factors in suppressing self-renewal or promoting differentiation of gastric epithelia. Notably, we found that Alk inhibits Wnt signaling by phosphorylating the tyrosine of Gsk3ß, while Bclaf3 and Prkra suppress regenerating islet-derived (Reg) genes by regulating the expression of epithelial interleukins. Therefore, Alk, Bclaf3, and Prkra may suppress stemness/proliferation and function as novel regulators of gastric epithelial differentiation.

Carbon Dioxide Fractional Laser Treatment Induces Lgr5+ Stem Cell Activation and Hair Regrowth Through the Canonical Wnt/ß-Catenin Pathway.

BACKGROUND: Different types of alopecia have negative impacts on patients. Recently, some kinds of laser or light therapies have been reported to effectively alleviate hair loss. Carbon dioxide fractional laser (CO2FL) treatment is one of the most effective laser treatments, but its beneficial effects and exact mechanism in hair regrowth have not been reported in detail. The purpose of this study was to investigate the effect and molecular mechanism further. METHODS: C57 and Lgr5-Cre: Rosa-mTmG mouse models of hair regrowth were established by CO2FL treatment, and the parameters that induced the best effect were determined. Tissues were harvested on the day prior to the treatment day and on days 3, 5, 7, 10 and 14 after CO2FL. H&E and immunofluorescence staining, RNA sequencing (RNA-seq), quantitative real-time polymerase chain reaction (qPCR), Western blotting (WB) and related inhibitor were used to determine the molecular mechanism underlying the effect of CO2FL treatment on the hair cycle and hair regrowth. In clinical trial, five participants were treated three sessions at 1-month intervals to obverse the effects. RESULTS: Hair regrew and covered the treatment area on the tenth day after CO2FL treatment with the best parameters, while the control group showed signs of hair growth on the 14th day. H&E and immunofluorescence staining showed that the transition of hair follicles (HFs) from telogen to anagen was accelerated, and the rapid activation and proliferation of Lgr5+ hair follicle stem cells (HFSCs) were observed in the treatment group. The RNA-seq, qPCR and WB results indicated that the Wnt pathway was significantly activated after CO2FL treatment. Improvement achieved with CO2FL treatment in clinical trial. CONCLUSIONS: The results of this study suggest that CO2FL treatment can promote hair regrowth by activating Lgr5+ HFSCs and upregulating the Wnt/ß-catenin pathway. Clinical trial results demonstrated that CO2FL treatment will be a promising therapeutic regimen for alopecia. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Age-Dependent Differences in Radiation-Induced DNA Damage Responses in Intestinal Stem Cells.

Age at exposure is a critical modifier of the risk of radiation-induced cancer. However, the effects of age on radiation-induced carcinogenesis remain poorly understood. In this study, we focused on tissue stem cells using Lgr5-eGFP-ires-CreERT2 mice to compare radiation-induced DNA damage responses between Lgr5+ and Lgr5- intestinal stem cells. Three-dimensional immunostaining analyses demonstrated that radiation induced apoptosis and the mitotic index more efficiently in adult Lgr5- stem cells than in adult Lgr5+ stem cells but not in infants, regardless of Lgr5 expression. Supporting this evidence, rapid and transient p53 activation occurred after irradiation in adult intestinal crypts but not in infants. RNA sequencing revealed greater variability in gene expression in adult Lgr5+ stem cells than in infant Lgr5+ stem cells after irradiation. Notably, the cell cycle and DNA repair pathways were more enriched in adult stem cells than in infant stem cells after irradiation. Our findings suggest that radiation-induced DNA damage responses in mouse intestinal crypts differ between infants and adults, potentially contributing to the age-dependent susceptibility to radiation carcinogenesis.

PTEN/AKT and Wnt/ß-catenin signaling pathways regulate the proliferation of Lgr5+ cells in liver cancer.

The progression and spread of tumors are believed to be primarily caused by cancer stem cells (CSCs). Nevertheless, the task of focusing on CSCs for cancer treatment continues to be difficult. Lgr5, a G-protein-coupled receptor containing leucine-rich repeats, is highly expressed in different types of cancer and serves as a distinctive marker for cancer stem cells (CSCs). In this study, we employed the Cre-loxP system and Lgr5 tracking mice of male to selectively remove PTEN and ß-catenin in Lgr5+ cells of DEN-induced liver cancer and monitor the behavior of Lgr5+ cells. The tracking data revealed that the activation of PTEN-mediated AKT signaling in Lgr5 led to a significant rise in the quantity of Lgr5+ cells, whereas the inhibition of Wnt/ß-catenin signaling decreased the number of cells in DEN-induced liver cancer. Therefore, we have shown that the growth of Lgr5+ cells can be controlled by the PTEN/AKT and Wnt/ß-catenin pathways, offering a potential treatment option for fighting against liver cancer.

Elevated Expression of LGR5 and WNT Signaling Factors in Neuroblastoma Cells With Acquired Drug Resistance.

Neuroblastoma (NB) is a pediatric solid cancer with high fatality, relapses, and acquired resistance to chemotherapy, that requires new therapeutic approaches to improve survival. LGR5 is a receptor that potentiates WNT/signaling pathway and has been reported to promote development and survival in several adult cancers. In this study we investigated LGR5 expression in a panel of NB cell lines with acquired resistance to vincristine or doxorubicin. We show LGR5-LRP6 cooperation with enhanced expression in drug resistant NB cell lines compared to parental cells, suggesting a role for LGR5 in the emergence of drug resistance, warranting further investigation.

Intestinal epithelial stem cell transplants as a novel therapy for cerebrovascular stroke.

Almost 2/3rds of stroke survivors exhibit vascular cognitive impairment and a third of stroke patients will develop dementia 1-3 years after stroke. These dire consequences underscore the need for effective stroke therapies. In addition to its damaging effects on the brain, stroke rapidly dysregulates the intestinal epithelium, resulting in elevated blood levels of inflammatory cytokines and toxic gut metabolites due to a 'leaky' gut. We tested whether repairing the gut via intestinal epithelial stem cell (IESC) transplants would also improve stroke recovery. Organoids containing IESCs derived from young rats transplanted into older rats after stroke were incorporated into the gut, restored stroke-induced gut dysmorphology and decreased gut permeability, and reduced circulating levels of endotoxin LPS and the inflammatory cytokine IL-17A. Remarkably, IESC transplants also improved stroke-induced acute (4d) sensory-motor disability and chronic (30d) cognitive-affective function. Moreover, IESCs from older animals displayed senescent features and were not therapeutic for stroke. These data underscore the gut as a critical therapeutic target for stroke and demonstrate the effectiveness of gut stem cell therapy.