肠道菌群：肠道干细胞增殖分化的调节者

肠道干细胞(intestinal stem cells, ISCs)是肠道各类上皮细胞的来源,通过平衡增殖与分化维持肠道稳态。同时,肠道菌群及其代谢物在维持宿主肠道稳态中也发挥着重要作用。随着技术的发展,研究者认识到ISCs与肠道菌群之间存在相互作用。研究表明,ISCs对上皮细胞亚型的调控影响肠道菌群的组成,并且肠道菌群及其代谢物也影响ISCs介导的上皮发育。本文阐述了ISCs分化对肠道菌群的影响,重点总结了肠道菌群及其代谢物调控ISCs增殖分化的研究进展,从菌群调控ISCs的角度探讨肠道损伤的治疗思路,并对未来可能的研究方向进行讨论。     

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.     

Current knowledge on the multiform reconstitution of intestinal stem cell niche

The development of “mini-guts” organoid originates from the identification of Lgr5⁺ intestinal stem cells (ISCs) and circumambient signalings within their specific niche at the crypt bottom. These in vitro self-renewing “mini-guts”, also named enteroids or colonoids, undergo perpetual proliferation and regulated differentiation, which results in a high-performance, self-assembling and physiological organoid platform in diverse areas of intestinal research and therapy. The triumphant reconstitution of ISC niche in vitro also relies on Matrigel, a heterogeneous sarcoma extract. Despite the promising prospect of organoids research, their expanding applications are hampered by the canonical culture pattern, which reveals limitations such as inaccessible lumen, confine scale, batch to batch variation and low reproducibility. The tumor-origin of Matrigel also raises biosafety concerns in clinical treatment. However, the convergence of breakthroughs in cellular biology and bioengineering contribute to multiform reconstitution of the ISC niche. Herein, we review the recent advances in the microfabrication of intestinal organoids on hydrogel systems.     

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the ‘‘stemness’’ of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.     

The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse

A frequent complication in colorectal cancer (CRC) is regeneration of the tumor after therapy. Here, we report that a gene signature specific for adult intestinal stem cells (ISCs) predicts disease relapse in CRC patients. ISCs are marked by high expression of the EphB2 receptor, which becomes gradually silenced as cells differentiate. Using EphB2 and the ISC marker Lgr5, we have FACS-purified and profiled mouse ISCs, crypt proliferative progenitors, and late transient amplifying cells to define a gene program specific for normal ISCs. Furthermore, we discovered that ISC-specific genes identify a stem-like cell population positioned at the bottom of tumor structures reminiscent of crypts. EphB2 sorted ISC-like tumor cells display robust tumor-initiating capacity in immunodeficient mice as well as long-term self-renewal potential. Taken together, our data suggest that the ISC program defines a cancer stem cell niche within colorectal tumors and plays a central role in CRC relapse.     

NAD+ supplementation rejuvenates aged gut adult stem cells

The tissue decline due to aging is associated with the deterioration of adult stem cell function. Here we show the number and proliferative activity of intestinal stem cells (ISCs) but not Paneth cells decline during aging, as does ISC function assessed ex vivo. Levels of SIRT1 and activity of mTORC1 also decline with aging. The treatment with the NAD(+) precursor nicotinamide riboside (NR) rejuvenates ISCs from aged mice and reverses an impaired ability to repair gut damage. The effect of NR is blocked by the mTORC1 inhibitor rapamycin or the SIRT1 inhibitor EX527. These findings demonstrate that small molecules affecting the NAD/SIRT1/mTORC1 axis may guide a translational path for maintenance of the intestine during aging.     

Microbial Metabolites and Intestinal Stem Cells Tune Intestinal Homeostasis

Microorganisms that colonize the gastrointestinal tract, collectively known as the gut microbiota, are known to produce small molecules and metabolites that significantly contribute to host intestinal development, functions, and homeostasis. Emerging insights from microbiome research reveal that gut microbiota‐derived signals and molecules influence another key player maintaining intestinal homeostasis—the intestinal stem cell niche, which regulates epithelial self‐renewal. In this review, the literature on gut microbiota‐host crosstalk is surveyed, highlighting the effects of gut microbial metabolites on intestinal stem cells. The production of various classes of metabolites, their actions on intestinal stem cells are discussed and, finally, how the production and function of metabolites are modulated by aging and dietary intake is commented upon.     

Paneth cells drive intestinal stem cell competition and clonality in aging and calorie restriction

Calorie restriction has been recently shown to increase intestinal stem cell competition and to reduce mutation fixation in young mice. However, the impact of aging on this process is unknown. By employing Confetti reporter mice, here we show that, unexpectedly, old mice have more intestinal stem cell (ISC) competition than young mice. Moreover, differently from what observed in young mice, calorie restriction, when applied at late-life, decreases this process. Importantly, we also observed a strong correlation between the ISC competition and Paneth cell number. In vivo analysis and in vitro organoid experiments indicated that Paneth cells play a major role in driving intestinal stem cell competition and crypt clonality. Taken together, our results provide evidence that increasing the number of Paneth cells can increase the number of competitive ISCs, representing a valuable therapeutic target to delay fixation of mutated intestinal stem cells.     

Intestinal stem cells in the adult Drosophila midgut

Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury.     

Casein kinase 1‐epsilon or 1‐delta required for Wnt‐mediated intestinal stem cell maintenance

The intestinal epithelium holds an immense regenerative capacity mobilized by intestinal stem cells (ISCs), much of it supported by Wnt pathway activation. Several unique regulatory mechanisms ensuring optimal levels of Wnt signaling have been recognized in ISCs. Here, we identify another Wnt signaling amplifier, CKIε, which is specifically upregulated in ISCs and is essential for ISC maintenance, especially in the absence of its close isoform CKIδ. Co‐ablation of CKIδ/ε in the mouse gut epithelium results in rapid ISC elimination, with subsequent growth arrest, crypt–villous shrinking, and rapid mouse death. Unexpectedly, Wnt activation is preserved in all CKIδ/ε‐deficient enterocyte populations, with the exception of Lgr5⁺ ISCs, which exhibit Dvl2‐dependent Wnt signaling attenuation. CKIδ/ε‐depleted gut organoids cease proliferating and die rapidly, yet survive and resume self‐renewal upon reconstitution of Dvl2 expression. Our study underscores a unique regulation mode of the Wnt pathway in ISCs, possibly providing new means of stem cell enrichment for regenerative medicine.     

Toll-like Receptor 4 Is Expressed on Intestinal Stem Cells and Regulates Their Proliferation and Apoptosis via the p53 Up-regulated Modulator of Apoptosis

Factors regulating the proliferation and apoptosis of intestinal stem cells (ISCs) remain incompletely understood. Because ISCs exist among microbial ligands, immune receptors such as toll-like receptor 4 (TLR4) could play a role. We now hypothesize that ISCs express TLR4 and that the activation of TLR4 directly on the intestinal stem cells regulates their ability to proliferate or to undergo apoptosis. Using flow cytometry and fluorescent in situ hybridization for the intestinal stem cell marker Lgr5, we demonstrate that TLR4 is expressed on the Lgr5-positive intestinal stem cells. TLR4 activation reduced proliferation and increased apoptosis in ISCs both in vivo and in ISC organoids, a finding not observed in mice lacking TLR4 in the Lgr5-positive ISCs, confirming the in vivo significance of this effect. To define molecular mechanisms involved, TLR4 inhibited ISC proliferation and increased apoptosis via the p53-up-regulated modulator of apoptosis (PUMA), as TLR4 did not affect crypt proliferation or apoptosis in organoids or mice lacking PUMA. In vivo effects of TLR4 on ISCs required TIR-domain-containing adapter-inducing interferon-β (TRIF) but were independent of myeloid-differentiation primary response-gene 88 (MYD88) and TNFα. Physiological relevance was suggested, as TLR4 activation in necrotizing enterocolitis led to reduced proliferation and increased apoptosis of the intestinal crypts in a manner that could be reversed by inhibition of PUMA, both globally or restricted to the intestinal epithelium. These findings illustrate that TLR4 is expressed on ISCs where it regulates their proliferation and apoptosis through activation of PUMA and that TLR4 regulation of ISCs contributes to the pathogenesis of necrotizing enterocolitis.     

EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells

Tissue-specific adult stem cells are commonly associated with local niche for their maintenance and function. In the adult Drosophila midgut, the surrounding visceral muscle maintains intestinal stem cells (ISCs) by stimulating Wingless (Wg) and JAK/STAT pathway activities, whereas cytokine production in mature enterocytes also induces ISC division and epithelial regeneration, especially in response to stress. Here we show that EGFR/Ras/ERK signaling is another important participant in promoting ISC maintenance and division in healthy intestine. The EGFR ligand Vein is specifically expressed in muscle cells and is important for ISC maintenance and proliferation. Two additional EGFR ligands, Spitz and Keren, function redundantly as possible autocrine signals to promote ISC maintenance and proliferation. Notably, over-activated EGFR signaling could partially replace Wg or JAK/STAT signaling for ISC maintenance and division, and vice versa. Moreover, although disrupting any single one of the three signaling pathways shows mild and progressive ISC loss over time, simultaneous disruption of them all leads to rapid and complete ISC elimination. Taken together, our data suggest that Drosophila midgut ISCs are maintained cooperatively by multiple signaling pathway activities and reinforce the notion that visceral muscle is a critical component of the ISC niche.     

Another notch in stem cell biology: Drosophila intestinal stem cells and the specification of cell fates

Previous work has suggested that many stem cells can be found in microanatomic niches, where adjacent somatic cells of the niche control the differentiation and proliferation states of their resident stem cells. Recently published work examining intestinal stem cells (ISCs) in the adult Drosophila midgut suggests a new paradigm where some stem cells actively control the cell fate decisions of their daughters. Here, we review recent literature((1)) demonstrating that, in the absence of a detectable stem cell niche, multipotent Drosophila ISCs modulate the Notch signaling pathway in their adjacent daughter cells in order to specify the differentiated lineages of their descendants. These observations made in Drosophila are challenging and advancing our understanding of stem cell biology.     

JAK/STAT signaling coordinates stem cell proliferation and multilineage differentiation in the Drosophila intestinal stem cell lineage

Adult stem cells are the most primitive cells of a lineage and are distinguished by the properties of self-renewal and multipotency. Coordinated control of stem cell proliferation and multilineage differentiation is essential to ensure a steady output of differentiated daughter cells necessary to maintain tissue homeostasis. However, little is known about the signals that coordinate stem cell proliferation and daughter cell differentiation. Here we investigate the role of the conserved JAK/STAT signaling pathway in the Drosophila intestinal stem cell (ISC) lineage. We show first, that JAK/STAT signaling is normally active in both ISCs and their newly formed daughters, but not in terminally differentiated enteroendocrine (ee) cells or enterocyte (EC) cells. Second, analysis of ISC lineages shows that JAK/STAT signaling is necessary but not sufficient for daughter cell differentiation, indicating that competence to undergo multilineage differentiation depends upon JAK/STAT. Finally, our analysis reveals JAK/STAT signaling to be a potent regulator of ISC proliferation, but not ISC self-renewal. On the basis of these findings, we suggest a model in which JAK/STAT signaling coordinates the processes of stem cell proliferation with the competence of daughter cells to undergo multilineage differentiation, ensuring a robust cellular output in the lineage.     

JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut

Metazoans employ cytoprotective and regenerative strategies to maintain tissue homeostasis. Understanding the coordination of these strategies is critical to developing accurate models for aging and associated diseases. Here we show that cytoprotective Jun N-terminal kinase (JNK) signaling influences regeneration in the Drosophila gut by directing proliferation of intestinal stem cells (ISCs). Interestingly, this function of JNK contributes to the loss of tissue homeostasis in old and stressed intestines by promoting the accumulation of misdifferentiated ISC daughter cells. Ectopic Delta/Notch signaling in these cells causes their abnormal differentiation but also limits JNK-induced proliferation. Protective JNK signaling and control of cell proliferation and differentiation by Delta/Notch signaling thus have to be carefully balanced to ensure tissue homeostasis. Our findings suggest that this balance is lost in old animals, increasing the potential for neoplastic transformation.