Expansion Of Intestinal Stem Cells Research Articles

L-Arginine Enhances Intestinal Stem Cell Function

Background: L-arginine preserves intestinal barrier integrity from injury, but underlying mechanisms remain unknown. Methods: In this study we utilized mice and small intestinal (SI) organoid models to clarify the role of L-arginine on epithelial differentiation of intestinal stem cells (ISCs). Findings: Here we show that L-arginine significantly increased expansion of ISCs, and Paneth cells in vivo and in vitro. Importantly, L-arginine stimulated ISC proliferation through Wnt/β-catenin signals of Paneth cells. Pre-treatment with L-arginine protected SI organoids in response to damage provoked by murine TNF-α and mice in response to gut injury provoked by a chemotherapy drug (5-FU). Interpretation: Our results demonstrate that L-arginine stimulated ISCs proliferation through Wnt/β-Catenin pathway activated by Wnt3a secreted by Paneth cells to protect the intestinal epithelial barrier. Funding Statement: This work was supported by National Key R&D Program of China (2018YFD0500600), the State Key Laboratory of Animal Nutrition (2004DA125184), National Natural Science Foundation of China (31502024), National Basic Research Program of China (973 program 2013CB127302), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Declaration of Interests: There are no conflicts of interest (financial, professional or personal) related to this manuscript. Ethics Approval Statement: All procedures were performed in accordance with institutional and national guidelines and regulations and were approved by the China Agricultural University Animal Care and Use Committee.

Unlimited expansion of intestinal stem cells from a wide range of ages.

The recent technical advance in cloning and culturing ground-state intestinal stem cells (ISC) provides us an opportunity of accurate assessment of age-related impact on the function of highly proliferative intestinal stem cells. Our ability of indefinitely and robustly expanding single-stem-cell derived pedigrees in vitro allows us to study intestinal stem cells at the clonal level. Interestingly, comparable number of ISC clones was yielded from 1mm endoscopic biopsy of all donors despite the age. They were passaged in vitro as pedigrees and expanded to 1 billion cells in approximately sixty days without changes in stemness demonstrated by clonogenicity and multipotency. Therefore, our study shows that ISCs from a wide range of ages can be cloned and expanded to unlimited number in vitro with similar efficiency and stability. These patient-derived ISCs harbor intrinsic immortality and are ideal for autologous transplantation, supporting the promise of adult-stem-cell based personalized medicine.

Supporting gut epithelial regeneration.

This study found that lactic-acid-producing bacteria support the expansion of intestinal stem cells and epithelial differentiation.

The mTORC1 signaling modulated by intracellular C3 activation in Paneth cells promotes intestinal epithelial regeneration during acute injury

Complement activation is associated with regional inflammation during acute gastrointestinal injury (AGI). This study is designed to explore how intracellular C3 activation in Paneth cells (PCs) affects regeneration of intestinal epithelium during AGI. AGI was induced in wildtype C57BL/6 mice, with sham operation employed as control. Exogenous C3 (1 mg, I.P.) was applied at 6 h post-surgery. Intestinal crypts harvested from ileum were cultured with presence or absence of C3 (20 μg/ml), with small interfering RNA against BST1 and complement activation inhibitor selectively applied in vitro. The intestinal integrity, percentage of PCs and intestinal stem cells (ISCs) were evaluated. Importantly, cADPR, C3 fragments, and S6-related proteins were detected in PCs to inspect the mammalian target of rapamycin complex 1 (mTORC1) signaling. AGI caused breakdown of intestinal mucosa integrity and regional inflammation. Exogenous C3 by itself failed to promote the growth of intestinal epithelium, but distinctly enhanced the activity of PCs via intracellular activation, which subsequently supported the expansion of ISCs inside of intestinal crypts. Inhibition of C3 activation was associated with decreased expressions of S6, S6K1 and cADPR, with blocking BST1 found to depress cADPR only. Collectively, these data confirmed intracellular activation of C3 in PCs enhanced expansion of ISCs in response to acute injury. The mTORC1 signaling pathway in PCs contributed to this crosstalk during exogenous C3 treatment.

IL22 Inhibits Epithelial Stem Cell Expansion in an Ileal Organoid Model

Background & AimsCrohn’s disease is an inflammatory bowel disease that affects the ileum and is associated with increased cytokines. Although interleukin (IL)6, IL17, IL21, and IL22 are increased in Crohn’s disease and are associated with disrupted epithelial regeneration, little is known about their effects on the intestinal stem cells (ISCs) that mediate tissue repair. We hypothesized that ILs may target ISCs and reduce ISC-driven epithelial renewal.MethodsA screen of IL6, IL17, IL21, or IL22 was performed on ileal mouse organoids. Computational modeling was used to predict microenvironment cytokine concentrations. Organoid size, survival, proliferation, and differentiation were characterized by morphometrics, quantitative reverse-transcription polymerase chain reaction, and immunostaining on whole organoids or isolated ISCs. ISC function was assayed using serial passaging to single cells followed by organoid quantification. Single-cell RNA sequencing was used to assess Il22ra1 expression patterns in ISCs and transit-amplifying (TA) progenitors. An IL22-transgenic mouse was used to confirm the impact of increased IL22 on proliferative cells in vivo.ResultsHigh IL22 levels caused decreased ileal organoid survival, however, resistant organoids grew larger and showed increased proliferation over controls. Il22ra1 was expressed on only a subset of ISCs and TA progenitors. IL22-treated ISCs did not show appreciable differentiation defects, but ISC biomarker expression and self-renewal–associated pathway activity was reduced and accompanied by an inhibition of ISC expansion. In vivo, chronically increased IL22 levels, similar to predicted microenvironment levels, showed increases in proliferative cells in the TA zone with no increase in ISCs.ConclusionsIncreased IL22 limits ISC expansion in favor of increased TA progenitor cell expansion.

Intestinal epithelial cell-specific IGF1 promotes the expansion of intestinal stem cells during epithelial regeneration and functions on the intestinal immune homeostasis.

It is well known that insulin-like growth factor 1 (IGF1) acts as a trophic factor in small intestine under both physiological and pathophysiological conditions. However, it still lacks direct in vivo evidence of the functions of intestinal epithelial cell (IEC)-specific IGF1 under both normal and pathological conditions. Using IEC-specific IGF1-knockout (cKO) mice and Lgr5-eGFP-CreERT mice, we demonstrate that IEC-specific IGF1 can enhance nutrient uptake, reduce protein catabolism and energy consumption, and promote the proliferation and expansion of intestinal epithelial cells, including intestinal epithelial stem cells and intestinal secretory cells. Next, we showed that IEC-specific IGF1 renders IECs resistant to irradiation and promotes epithelial regeneration. Strikingly, transcriptome profiling assay revealed that many differentially expressed genes involved in the differentiation and maturation of lymphoid lineages were significantly suppressed in the cKO mice as compared with the control mice. We demonstrated that deletion of IGF1 in IECs enhances bacterial translocation to the mesenteric lymph nodes and liver. Furthermore, high-throughput sequencing of 16S ribosomal RNA genes of gut microbiota revealed that IEC-specific IGF1 loss profoundly affected the gut microbial composition at various levels of classification. Therefore, our findings shed light on the in vivo roles of IEC-specific IGF1 in intestinal homeostasis, epithelial regeneration, and immunity, broadening our current insights on IGF1 functions.

Synthesis and characterization of well-defined hydrogel matrices and their application to intestinal stem cell and organoid culture.

Growing cells within an extracellular matrix-like 3D gel is required for, or can improve, the growth of many cell types ex vivo. Here, we describe a protocol for the generation of well-defined matrices for the culture of intestinal stem cells (ISCs) and intestinal organoids. These matrices comprise a poly(ethylene glycol) (PEG) hydrogel backbone functionalized with minimal adhesion cues including RGD (Arg-Gly-Asp), which is sufficient for ISC expansion, and laminin-111, which is required for organoid formation. As such, the hydrogels present a defined and reproducible, but also tunable, environment, allowing researches to manipulate physical and chemical parameters, and examine their influence on ISC and organoid growth. Hydrogels are formed by an enzymatic cross-linking reaction of multiarm PEG precursors bearing glutamine- and lysine-containing peptides. PEG precursors containing either stable or hydrolytically degradable moieties are used to produce mechanically softening hydrogels, which are used for the expansion of ISCs or the formation of organoids, respectively. We also provide protocols for immunofluorescence analysis of cellular structures grown within these matrices, as well as for their dissociation and retrieval of cells for downstream use. Hydrogel precursors can be produced and their mechanical properties characterized to ascertain stiffness within 5-7 d. Hydrogel formation for ISC expansion or organoid formation takes 1-2 h. The materials described here can be readily adapted for the culture of other types of normal or transformed organoid structures.

79 - TLR4-Driven Tumorigenesis is Associated with an Expansion of Intestinal Stem Cells

79 - TLR4-Driven Tumorigenesis is Associated with an Expansion of Intestinal Stem Cells

Injury-stimulated and self-restrained BMP signaling dynamically regulates stem cell pool size during Drosophila midgut regeneration

Many adult organs rely on resident stem cells to maintain homeostasis. Upon injury, stem cells increase proliferation, followed by lineage differentiation to replenish damaged cells. Whether stem cells also change division mode to transiently increase their population size as part of a regenerative program and, if so, what the underlying mechanism is have remained largely unexplored. Here we show that injury stimulates the production of two bone morphogenetic protein (BMP) ligands, Dpp and Gbb, which drive an expansion of intestinal stem cells (ISCs) by promoting their symmetric self-renewing division in Drosophila adult midgut. We find that BMP production in enterocytes is inhibited by BMP signaling itself, and that BMP autoinhibition is required for resetting ISC pool size to the homeostatic level after tissue repair. Our study suggests that dynamic BMP signaling controls ISC population size during midgut regeneration and reveals mechanisms that precisely control stem cell number in response to tissue needs.

Well-defined matrices for the expansion of intestinal stem cells and organoids

Well-defined matrices for the expansion of intestinal stem cells and organoids

Single cell lineage tracing reveals a role for TgfβR2 in intestinal stem cell dynamics and differentiation

Intestinal stem cells (ISCs) are maintained by a niche mechanism, in which multiple ISCs undergo differential fates where a single ISC clone ultimately occupies the niche. Importantly, mutations continually accumulate within ISCs creating a potential competitive niche environment. Here we use single cell lineage tracing following stochastic transforming growth factor β receptor 2 (TgfβR2) mutation to show cell autonomous effects of TgfβR2 loss on ISC clonal dynamics and differentiation. Specifically, TgfβR2 mutation in ISCs increased clone survival while lengthening times to monoclonality, suggesting that Tgfβ signaling controls both ISC clone extinction and expansion, independent of proliferation. In addition, TgfβR2 loss in vivo reduced crypt fission, irradiation-induced crypt regeneration, and differentiation toward Paneth cells. Finally, altered Tgfβ signaling in cultured mouse and human enteroids supports further the in vivo data and reveals a critical role for Tgfβ signaling in generating precursor secretory cells. Overall, our data reveal a key role for Tgfβ signaling in regulating ISCs clonal dynamics and differentiation, with implications for cancer, tissue regeneration, and inflammation.

Concise Review: The Potential Use of Intestinal Stem Cells to Treat Patients with Intestinal Failure.

Intestinal failure is a rare life‐threatening condition that results in the inability to maintain normal growth and hydration status by enteral nutrition alone. Although parenteral nutrition and whole organ allogeneic transplantation have improved the survival of these patients, current therapies are associated with a high risk for morbidity and mortality. Development of methods to propagate adult human intestinal stem cells (ISCs) and pluripotent stem cells raises the possibility of using stem cell‐based therapy for patients with monogenic and polygenic forms of intestinal failure. Organoids have demonstrated the capacity to proliferate indefinitely and differentiate into the various cellular lineages of the gut. Genome‐editing techniques, including the overexpression of the corrected form of the defective gene, or the use of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 to selectively correct the monogenic disease‐causing variant within the stem cell, make autologous ISC transplantation a feasible approach. However, numerous techniques still need to be further optimized, including more robust ex vivo ISC expansion, native ISC ablation, and engraftment protocols. Large‐animal models can to be used to develop such techniques and protocols and to establish the safety of autologous ISC transplantation because outcomes in such models can be extrapolated more readily to humans. Stem Cells Translational Medicine 2017;6:666–676

AIMP2 Controls Intestinal Stem Cell Compartments and Tumorigenesis by Modulating Wnt/β-Catenin Signaling.

Wnt/β-catenin (CTNNB1) signaling is crucial for the proliferation and maintenance of intestinal stem cells (ISC), but excessive activation leads to ISC expansion and eventually colorectal cancer. Thus, negative regulators are required to maintain optimal levels of Wnt/β-catenin signaling. Aminoacyl-tRNA synthetase-interacting multifunctional proteins (AIMP) function in protein synthesis, but have also been implicated in signaling cascades affecting angiogenesis, immunity, and apoptosis. In this study, we investigated the relationship between AIMP2 and Wnt/β-catenin signaling in a murine model of intestinal homeostasis and tumorigenesis. Hemizygous deletion of Aimp2 resulted in enhanced Wnt/β-catenin signaling, increased proliferation of cryptic epithelial cells, and expansion of ISC compartments. In an Apc(Min/+) background, Aimp2 hemizygosity increased adenoma formation. Mechanistically, AIMP2 disrupted the interaction between AXIN and Dishevelled-1 (DVL1) to inhibit Wnt/β-catenin signaling by competing with AXIN. Furthermore, AIMP2 inhibited intestinal organoid formation and growth by suppressing Wnt/β-catenin signaling in an Aimp2 gene dosage-dependent manner. Collectively, our results showed that AIMP2 acts as a haploinsufficient tumor suppressor that fine-tunes Wnt/β-catenin signaling in the intestine, illuminating the regulation of ISC abundance and activity. Cancer Res; 76(15); 4559-68. ©2016 AACR.

MTORC1 and SIRT1 Cooperate to Foster Expansion of Gut Adult Stem Cells during Calorie Restriction

Longevity-promoting caloric restriction is thought to trigger downregulation of mammalian target of rapamycin complex 1 (mTORC1) signaling and upregulation of SIRT1 activity with associated health benefits. Here, we show that mTORC1 signaling in intestinal stem cells (ISCs) is instead upregulated during calorie restriction (CR). SIRT1 deacetylates S6K1, thereby enhancing its phosphorylation by mTORC1, which leads to an increase in protein synthesis and an increase in ISC number. Paneth cells in the ISC niche secrete cyclic ADP ribose that triggers SIRT1 activity and mTORC1 signaling in neighboring ISCs. Notably, the mTOR inhibitor rapamycin, previously reported to mimic effects of CR, abolishes this expansion of ISCs. We suggest that Paneth cell signaling overrides any direct nutrient sensing in ISCs to sculpt the observed response to CR. Moreover, drugs that modulate pathways important in CR may exert opposing effects on different cell types.

Fully defined matrices for the culture and expansion of intestinal stem cells and organoids

Fully defined matrices for the culture and expansion of intestinal stem cells and organoids

Intestinal stem cells and stem cell-based therapy for intestinal diseases.

Currently, many gastrointestinal diseases are a major reason for the increased mortality rate of children and adults every year. Additionally, these patients may cope with the high cost of the parenteral nutrition (PN), which aids in the long-term survival of the patients. Other treatment options include surgical lengthening, which is not sufficient in many cases, and intestinal transplantation. However, intestinal transplantation is still accompanied by many challenges, including immune rejection and donor availability, which may limit the transplant's success. The development of more safe and promising alternative treatments for intestinal diseases is still ongoing. Stem cell-based therapy (SCT) and tissue engineering (TE) appear to be the next promising choices for the regeneration of the damaged intestine. However, suitable stem cell source is required for the SCT and TE process. Thus, in this review we discuss how intestinal stem cells (ISCs) are a promising cell source for small intestine diseases. We will also discuss the different markers were used to identify ISCs. Moreover, we discuss the dominant Wnt signaling pathway in the ISC niche and its involvement in some intestinal diseases. Additionally, we discuss ISC culture and expansion, which are critical to providing enough cells for SCT and TE. Finally, we conclude and recommend that ISC isolation, culture and expansion should be considered when SCT is a treatment option for intestinal disorders. Therefore, we believe that ISCs should be considered a cell source for SCT for many gastrointestinal diseases and should be highlighted in future clinical applications.

Details Unfold: The Endoplasmic Reticulum Stress Response in Intestinal Inflammation and Cancer

Details Unfold: The Endoplasmic Reticulum Stress Response in Intestinal Inflammation and Cancer

A serum-free medium developed for in vitro expansion of murine intestinal stem cells.

Intestinal stem cells (ISCs) are located at the base of the intestinal crypts and have the ability to self-renew as well as to differentiate into mature epithelial cells. Recently, ISCs have received much attention for the treatment of many intestinal diseases. However, many challenges face those studying ISCs because insufficient ISCs are available. Therefore, the development of a culture medium for ISC expansion is an important necessity for basic research and clinical application. In this study, we described the technique used to develop a serum-free medium for expanding ISCs in vitro. Furthermore, five serum substitutes were selected and optimized in order to maintain the long-term proliferation and enteroid-forming ability of ISCs: (i) ethanolamine; (ii) ascorbic acid phosphate; (iii) transferrin; (iv) glutathione; and (v) sodium selenite. Analysis of gene expression of Lgr5, Bmi1, Msi1 and PTEN demonstrated that our serum-free medium sustained the expression of genes involved in ISC-related functions in the expanded ISCs. Additionally, the expression intensity of surface markers, including Lgr5, CD24 and CD44, on serum-free expanded cells in crypts was greatly increased. Taken together, our results demonstrate that the number of ISCs can be expanded and their functionality maintained in our serum-free medium, indicating the suitability of this serum-free expansion medium for increasing the numbers of ICSs available for basic research and clinical applications in the future.

Small intestinal stem cells

To summarize our current understanding of small intestinal stem cell biology and the current tools available for studying intestinal stem cells (ISCs). Recent reviews and original reports point toward the presence of two distinct populations of stem cells (ISCs) within the intestinal crypts. Until recently, the study of these two populations has been hindered by the lack of biomarkers available for isolation and characterization of ISCs and the absence of suitable culture conditions for expansion of ISCs in vitro. With the accumulation of various surface markers and transgenic mouse models, we have been able to gain a better understanding of the genetic signature of ISCs. In addition, these tools have provided opportunities to begin to study how ISCs are influenced by the various components of the ISC niche, including fibroblasts, bacteria, lymphoid cells, and Paneth cells. Advances in culture conditions now allow for the establishment of in-vitro studies of ISC function and dynamics. This brief review provides a general historical perspective of our understanding of the delineation of the two ISC populations. Furthermore, it discusses the known ISC markers and how these markers have been used to isolate and characterize ISC populations.

Type of Perioperative Diet Affects Long Term Adaptation Following Ileocecal Resection (ICR)

Our previous studies have demonstrated that expansion of intestinal stem cells (ISCs) results in crypt fission (CF) leading to sustained increases in intestinal surface area following massive small bowel loss. Based on this work, we hypothesize that therapies, including types of diet, that augment this response would result in sustained adaptation beyond the period of treatment due to the persistent increase in overall crypts. In this study, we sought to determine if the administration of a semi-elemental diet for a defined perioperative period would increase the expansion of ISCs and CF, resulting in sustained adaptive response.