Sarcomas are uncommon malignancies of mesenchymal origin that can arise throughout the human lifespan, at any part of the body. Surgery remains the optimal treatment modality whilst response to conventional treatments, such as chemotherapy and radiation, is minimal. Immunotherapy has emerged as a novel approach to treat different cancer types but efficacy in soft tissue sarcoma and bone sarcoma is limited to distinct subtypes. Growing evidence shows that cancer-stroma cell interactions and their microenvironment play a key role in the effectiveness of immunotherapy. However, the pathophysiological and immunological properties of the sarcoma tumor microenvironment in relation to immunotherapy advances, has not been broadly reviewed. Here, we provide an up-to-date overview of the different immunotherapy modalities as potential treatments for sarcoma, identify barriers posed by the sarcoma microenvironment to immunotherapy, highlight their relevance for impeding effectiveness, and suggest mechanisms to overcome these barriers. Sarcomas are a group of rare and highly heterogeneous malignancies encompassing more than 100 distinct histological subtypes Despite recent advances in cancer research, developments in sarcoma treatment have been slow. For localized disease, the primary treatment option is surgical resection of the tumor with adjuvant or neoadjuvant radiation therapy in selected cases. Localized soft tissue sarcoma and bone sarcoma patients have a 5-year disease free survival rate >80 % and 70 %, respectively. However, disease relapse occurs in more than half of the patients, often with the development of distant metastasis. Advanced disease cases are extremely challenging to treat. Conventional chemotherapeutics do not lead to durable responses or cure and patients may experience substantial toxicities. The standard therapy for metastatic disease is primarily structured around anthracycline-based chemotherapy in situ vaccination (iSV) including oncolytic virotherapy. Indeed, there is a large number of clinical trials for the use of immunotherapy in patients with sarcoma, but none of them has led to approval yet. The absence of dramatic immunotherapeutic responses in most cases has been attributed to a variety of factors, including barriers imposed by the tumor microenvironment that among others hinder the delivery of the immunotherapeutic agents and cause immunosuppression. The current review i) summarizes current knowledge on immunotherapy application in soft tissue and bone sarcoma in the clinical and preclinical setting over the last decade, ii) discusses the barriers posed by the sarcoma microenvironment hindering immunotherapy efficacy and iii) provides an overview of potential strategies that are tailored to overcome these barriers.A flurry of new research is now exploring the role of immunotherapy in sarcoma. Unlike chemotherapy, which directly kills cancer cells, immunotherapy relies on stimulating the natural defenses of the host immune system to attack malignant cells. Modalities of immunotherapy can be grouped in the following clusters: immune checkpoint inhibitors (ICIs), adoptive cell therapy (ACT), cancer vaccines and Immune checkpoints have evolved to act as gatekeepers of immune responses by suppressing inflammation-induced tissue damage and autoimmunity. However, cancer cells hijack the immune checkpoint signaling by upregulating inhibitory immunoreceptors  on immune cell surface, capable of suppressing both antigen and co-stimulatory signaling upon ligand engagement With regards to sarcoma patients, blockade of the PD-1/PD-L1 axis has been promising for specific histological subtypes. A completed phase 2 clinical trial, SARC028  and (ii) ACT with genetically engineered T cell receptor (TCR) or synthetic chimeric antigen receptor (CAR) targeting tumor specific antigens ACT, also known as cellular immunotherapy, comprises the intravenous transfer of either tumor-resident or genetically modified blood-derived immune cells into patients to augment antitumor immune responses. The most widespread form of ACT is T cell based and can be classified into (i) ACT with + TAMs, compared to the pre-infusion state With respect to TCR-modified cell therapy, an affinity-enhanced TCR recognizing the cancer testis antigen, NY-ESO-1, reported encouraging results for the treatment of metastatic synovial sarcoma  combined with adjuvants. The ultimate goal is to provoke an adaptive T cell response capable of eradicating residual tumor and establishing a lasting antitumor memory in the absence of adverse effects and non-specific reactions. Traditionally, antigens can be delivered in the form of DNA, RNA and peptide itself, or via autologous dendritic cells (DCs). Administration of autologous DCs matured with autologous tumor lysate and keyhole limpet hemocyanin was found to improve clinical outcome in patients diagnosed with Ewing/rhabdomyosarcoma, whereas other histological subtypes failed to respond. Of note, T cell responses to autologous tumor lysate were identified in more than half of immunotherapy recipients, in agreement with a higher survival, while an enhanced immune reconstitution was reported after addition of interleukin 7 (IL7) iSVs comprise a novel arm of cancer immunotherapy. As opposed to conventional vaccines, iSVs are antigen-agnostic agents having the ability to mount endogenous antitumor responses, directly or indirectly, by “generating” a vaccine within the TME following sourcing of antigens from dead or dying tumor cells. The mechanisms of action of iSVs mainly rely on the activation of innate immune pattern recognition receptors  and stimulator of interferon genes (STING) protein oncolytic viruses. Genetically or chemically modified oncolytic viruses expressing immunomodulators specifically infect and replicate within cancer cells inducing immunogenic cell death and consequent release of TAA and neoantigens. This process results in local priming of the immune system (recruitment of DCs and NK cells to the tumor site), leading to an effective rejection of both virus-injected and distant tumors Another class of recently introduced iSV agents are the Despite the rapid advances made in the field, immunotherapy for sarcoma treatment is still in its infancy. The rarity, heterogeneity and complexity of these mesenchymal malignancies create challenges around diagnosis and treatment decisions. Thus, significant attention should be paid during trial design and interpretation of results. An approach to improve clinical trial design and eliminate uncertainties about the treatment effect in such rare diseases is the consideration of comprehensive preclinical evidence that strongly supports the effectiveness of a particular therapeutic intervention for a specific sarcoma subtype.Fibrosarcoma is the most frequently employed tumor model in the preclinical setting with regards to soft tissue sarcoma immunotherapy . Nevertheless, emerging preclinical work reported that targeting of FZD10 combined with radioimmunotherapy exhibited a potent antitumor activity against xenograft tumors. Specifically, it was shown that labeling of anti-FZD10 antibody with α-emitting radionuclides is superior to β-emitters labeling with no apparent systemic toxicities. FZD10 is highly expressed in synovial sarcoma and serves as a putative receptor of Wnt signaling and thus, a promising target for iSV osteosarcoma treatment in clinical practice. Although the clinical benefit from ICI alone is rather sporadic, preclinical studies indicate that combinations with other modalities of immunotherapy can significantly potentiate their antitumor responses . The glycosphingolipid disialoganglioside GD2 is a well-known TAA implicated in tumor cell proliferation and currently explored in neuroblastoma, melanoma and sarcoma clinical trials  In vitro evidence indicated that co-culture of MeV-infected sarcoma cells with NK cells stimulated the release of GZMA/B, perforin and granulysin resulting in higher oncolysis rates when compared to the respective monotherapies The preclinical research activity for chondrosarcoma is restricted to a single study. Consistent with the clinical application of zoledronate (ZOL), a nitrogen-containing bisphosphonate sensitizing tumors to Vγ9Vδ2 T cell-mediated cytotoxicity, Sun et al. showed that weekly intravenous ZOL administration improved Vγ9Vδ2 T cell cytotoxic in a TCR-dependent and via perforin-mediated mechanisms, resulting in potent antitumor effects Unlike osteosarcoma and Ewing sarcoma, preclinical evaluation of immunotherapy in The intrinsically heterogeneous nature of sarcomas and the complexity of the TME have a decisive role in their behavior and response to treatment. In the TME, tumor cells coexist with heterotypic cell populations including immune cells, endothelial cells, pericytes, cancer associated fibroblasts (CAFs), mesenchymal stem cells (MSCs) and nerve fibers, with which they communicate via direct cellular contact and an array of paracrine signals. Communication between cancer cells and stroma is also modulated by extracellular matrix components and the microbiome.+ T lymphocytes in the TME, solid tumors are generally classified into highly inflamed- “hot” and non-inflamed- “cold”. “Cold” tumors can be further subdivided into immune desert or immune excluded  in tumor cells and various types of immune cells, such as MDSCs, macrophages, DCs, and bone marrow-derived macrophages + T cells and IFN production by Th1 cells. Hypoxia may also upregulate PD-1, CTLA-4 and TGF-β expression on the surface of T cells through the adenosine (Ado-A2aR) pathway which further promotes immune tolerance by diverting the cytokine and cellular profile of the TME away from cytotoxic T cell inflammation, leading to tumor progression and metastasis In agreement with other solid malignancies, sarcomas are defined by a leaky and fragile vascularture which impedes proper tissue oxygenation and nutrient delivery. To compensate for nutrient and oxygen scarcity, tumor cells trigger the HIF signaling pathway and consequent expression of pro-angiogenic proteins, such as vascular endothelial growth factor (VEGF), angiopoietin-1/-2, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF). HIF-1α is the transcriptional activator of CTLA-4 in CD8In addition to the aberrant genomic instability caused by alterations in DNA repair pathways and release of free radicals, hypoxia promotes tumor aggressiveness via transcriptional regulation of downstream targets that sustain tumor aggressiveness and low immune cell infiltration The hypoxic stroma might be also exploited to activate prodrugs. For example, the hypoxia-activated prodrug TH-302 was found to significantly reduce hypoxia in a preclinical mouse prostate model while its combination with ICIs cured more than 80% of tumors by restoring T cell infiltration and reducing MDSCs CAFs are the major source of collagen synthesis and most abundant cell population in the TME. They may coexist as a heterogeneous population characterized by distinct phenotypic markers, gene expression profile and functionality + T cells to the TME, while on the other, they release IL17 inducing the production of angiogenic factors from fibroblasts and cancer cells. Moreover, the direct interaction with T cells via the cell surface ligands displayed by CAFs prevents their trafficking within the TME. Adding to this, MHC-I-antigen presentation by CAFs combined with PD-L2 and FASL expression on the CAF cell surface, can result in killing of antigen-specific cytotoxic CD8+ T cells.Another barrier of the TME that restricts infiltration of lymphocytes is the strong expression of mesenchymal and collagen barrier molecules in the ECM. In tumors, the ECM can undergo structural rearrangements to support tumor growth, including the production of collagen-driven fibrosis, a hallmark of many desmoplastic tumors Unlike epithelial cancers, the current understanding of the origin and contribution of CAFs in sarcomas is very limited. Studies in Ewing sarcoma in vitro findings demonstrate that MSCs stimulate osteosarcoma cells to express pro-angiogenic factors that promote the formation endothelial capillaries. Of course, MSCs have distinct immunomodulatory properties. Various studies have reported that they may sustain tumor progression by inhibiting T cell proliferation and activation, suppressing the cytotoxic activity of NKs, redirecting macrophages towards the immunosuppressive M2 phenotype and promoting regulatory T cell differentiation. In addition, MSCs inhibit B cell proliferation and antibody production While there is not a single cell origin ascribed to all sarcomas, increasing evidence suggests that MSCs are the sarcoma-initiating cells. MSCs are multipotent stem cells that differentiate towards diverse cell types including adipocytes, osteocytes, neural cells, fibroblast, chondrocytes and skeletal myoblasts. Several studies suggest that sarcomas arise from the malignant transformation of primitive MSCs or progenitor cells and many share a similar gene signature with the differentiated state of MSCs, possibly explaining the heterogeneity of these cancers. Although MSCs correspond to a small fraction of cells within the TME, yet they have a critical role in shaping the TME by promoting stemness, epithelial to mesenchymal transition, enhancing aggressiveness and drug resistance. It is very likely that resident tumor MSCs are responsible for relapse and metastasis. In osteosarcoma, the acidic microenvironment activates MSCs by inducing clonogenicity and invasion + T cells and NK cells. Furthermore, lactic acid inhibits monocyte activation and DC differentiation, promotes M2-polarization via increased arginase and HIF1α stabilization and increases the number of MDSCs. Finally, it may enhance the survival of Tregs, given the ability of Tregs to metabolize oxidized exogenous lactate. Many pediatric tumors including osteosarcoma and Ewing sarcoma have upregulated glycolysis and lactic acid fermentation. In fact, the oncogenic fusion protein EWS/FLI1 present in 80% of Ewing sarcoma cases, is the key regulator of the aberrant glycolytic reprograming of cancer cells Stroma acidification is another essential aspect of sarcoma microenvironment impeding immunotherapy. The limited delivery of serum nutrients and hypoxia push cancer cells to adopt alternative metabolic routes to cope with the high energetic demand for proliferation and survival. The catabolism of glucose via aerobic glycolysis is one primary metabolic adaptation that cancer cells undertake. However, a significant fraction of pyruvate is converted into lactic acid and secreted from the cell. Accumulation of lactic acid and other metabolic waste products in the TME impairs the function of immune cells inhibiting T cell proliferation and IFNγ production and thus, compromising the effector function of CD8Depending on the cell type and environmental conditions the products of glycolysis can be utilized to produce nucleotides or enter the mitochondrial tricarboxylic acid cycle (TCA) cycle which in turn will provide the metabolic intermediates required for biosynthesis of lipids and amino acids. However, decreased entry of glucose into the TCA cycle combined with the shuttling of intermediates into the biosynthetic pathways, imposes a need for TCA anaplerosis with an alternative carbon source other than glucose. The most common carbon source involved in TCA anaplerosis is the glutamine.Unlike glucose, which is required for both cancer and immune cell growth, glutamine is differentially utilized by each of these populations. Interestingly, inflammatory anti-tumor immune cells like M1 macrophages have less dependency on glutamine metabolism compared to M2 which rely on glutaminolysis for expansion. Also, studies in undifferentiated pleomorphic sarcoma showed that these cancer cells largely rely on glutamine as a source of energy and biosynthetic anabolism. Targeting of glutamine metabolism has been recently explored in preclinical models of undifferentiated pleomorphic sarcoma and additional soft tissue sarcoma subtypes showing encouraging results + T cells benefit from L-arginine uptake by enhancing survival, memory formation and anti-tumor efficacy. Different primary sarcomas like osteosarcoma, Ewing sarcoma and rhabdomyosarcoma were reported to have lost or lack the ability to synthesize arginine de novo (arginine auxotrophic) and highly depend on extracellular arginine in TME In addition to glutamine, cancer cells consume large quantities of arginine and tryptophan amino acids to support their growth and promote immune tolerance. Arginine is a crucial conditional amino acid for both cancer and immune cells. Arginine metabolism relies on the activity of arginase and inducible nitric oxide synthase (iNOS). Lactic acid abundance in TME favors the catabolism of arginine in myeloid cells via the arginase over iNOS, resulting in increased secretion of tumor-supporting factors by TAMs. On the contrary, TAMs utilizing iNOS exhibit an M1 phenotype and their nitric oxide upregulates the expression of VCAM1 adhesion molecules and subsequent T cell extravasation and homing against tumors. Importantly, CD8As opposed to glutamine and arginine, tryptophan is an essential amino acid which must be taken from the diet. Tryptophan acts as a substrate for kynurenine pathway. In tumors, however, increased levels of indoleamine 2,3-dioxygenase (IDO) catabolic enzymes  deplete tryptophan from TME and promote the production of immunosuppressive kynurenine metabolites. Increased IDO1 activity prevents activation of effector T cells, inhibits NK function and supports Treg differentiation and MDSCs infiltration. Despite clinical trials using IDO inhibitors have so far led to disappointing results, combinations with anti-PD-1 improved the objective response rates in melanoma patients. Given the fact that IDO1 is highly expressed on Ewing sarcoma cells and has been associated with worse outcome in osteosarcoma, among other cancers We have thus far discussed the current progress in immunotherapy for the treatment of soft tissue and bone sarcoma with emphasis on the barriers impeding its antitumor activities. It appears that the high metabolic rate of cancer cells in conjunction with the poor vascularization and the limited nutrient exchange lead to a fierce competition for resources. In a TME deprived from amino acids and glucose, T cells fail to substantially increase their nutrient or engage the appropriate metabolic pathway, required to mount proper immune responses. In an effort to overcome these challenges, research has in part focused on reprogramming the TME as a promising approach for increasing the efficacy of many therapeutic agents, ranging from standard chemotherapeutics to nanomedicine and immunotherapy modalities. TME can be reprogrammed  both at the vascular and stromal level, so that it morphologically and functionally resembles the non-malignant state of the tissue Nevertheless, application of antiangiogenic drugs does not impact perfusion of tumors with extremely compressed vessels, such as many sarcoma and pancreatic adenocarcinoma types, consistent with poor clinical response mechanotherapeutics” Molecules with tissue reprogramming capabilities are known as “+ cells could decrease the frequencies of intILC1 and ILC1 populations, which have been associated with immune suppression, in fibrosarcoma TME Another mechanotherapeutic approach holding great promise in potentiating immunotherapy is the targeting of TGFβ signaling pathway. TGFβ signaling is abnormally upregulated in most cancers and widely associated with tumor growth and progression. TGFβ is expressed by cancer cells, DCs, macrophages, CAFs and immature myeloid cells, indicating functional divergence. Its role in CAFs has been linked to immunosuppressive responses via attenuation of tumor response to PD-L1 blockade and contributing to T cell exclusion ® and Doxil® - liposomal formulations of daunorubicin and doxorubicin, respectively - were the first nanomedicines to be approved for the treatment of HIV-related Kaposi's sarcoma ®), a nanoparticle albumin-bound paclitaxel, which is a new type of taxane antineoplastic drug and has been indicated for the treatment of metastatic breast cancer, non-small cell lung cancer and metastatic pancreatic cancer Nanomedicine has improved therapeutic index and quality of life of sarcoma patients. DaunoXome® in mice bearing fibrosarcoma tumors yields improved anti-tumor effects than the conventional maximum tolerated dose schedule ® normalizes the TME, making the tumors softer, increasing perfusion and reducing interstitial fluid pressure and thus, overcoming the patho-physiological barriers to nanoparticle delivery. As a result of these normalization effects, combination of ICI with Doxil® can revert immunosuppression and improve ICI efficacy An emerging strategy to optimize the benefits of the EPR effect is by normalizing the TME, which results in reduction of interstitial fluid pressure without affecting tumor vessel hyperpermeability. With respect to this, we have shown that administration of nanomedicines in a metronomic fashion, defined as the frequent, low dose administration compared to the maximum tolerated dose at long time intervals, can normalize the TME. Although not clinically approved yet, our preclinical findings demonstrate that the metronomic administration of DoxilNanoparticle formulations could be further employed to normalize the TME by encapsulation of mechanotherapeutic agents that preferentially target the tumor. In this regard, nanomedicines designed to simultaneously target components of the TME and kill cancer cells can be used to broaden the therapeutic window of anticancer drugs. The development of drug delivery systems incorporating angiotensin receptor blockers as mechanotherapeutics conjugated to polymers that can be selectively degraded in the TME upon exposure to low pH is such an example Several non-invasive imaging modalities can be employed for monitoring intratumoral drug distribution and efficacy, such as ultrasound imaging, magnetic resonance imaging (MRI), computed tomography (CT), Positron Emission Tomography (PET), single photon emission computed tomography (SPECT) and optical imaging ® with the PD-1 inhibitor atezolizumab has already been demonstrated in a phase 3 clinical trial for the treatment of triple negative breast cancer and in a retrospective study of soft tissue sarcoma, with angiosarcoma patients reporting a significantly prolonged free survival compared to other subtypes ®, similarly to Abraxane®, induces immunogenic cell death by promoting immune cell infiltration and reverting tumor immunosuppression Combining nanomedicine with immunotherapy aims to reinforce key steps of the cancer immunity cycle Figure . The cliImmunotherapy advances, despite their promise, are often associated with immune related adverse events (irAEs), as a consequence of the nonspecific immune activation in the human body. irAEs differ from the classical chemotherapy-induced toxicities and may occur in almost any organ such as colon, muscle, lungs, liver and thyroid. Their frequency depends on the dosage, regimens and exposure time but also on patient's intrinsic factors. A few studies have specifically assessed the safety of ICI in patients with recurrent or therapy refractory sarcomas. A phase 1 dose escalation study (NCT01445379), investigating the tolerance and toxicity profile of ipilimumab monotherapy in children and young adults indicated that the occurrence of high grade irAEs associates with better response to CTLA-4 inhibition, although a proportional increase in the frequency of irAEs was observed with an increase in dose level. A different study assessing the safety of nivolumab as monotherapy  showed that administration of 3 mg/kg every 14 days is well tolerated in children with Ewing, rhabdomyosarcoma, and osteosarcoma subtypes and the most common irAEs were: increased lipase levels and cardiac and pleural effusion. In the SARC028 (NCT02301039) study, the most frequent grade 3 or worse adverse effect reported in both bone and soft tissue sarcoma groups was anaemia, followed by decreased lymphocyte count and prolonged activated partial thromboplastin time. Also, some patients of the bone sarcoma group had decreased platelet count. Notably, none of these treatment-related serious adverse events were fatal. Alliance  In general, the incidence of fatal irAEs for ICI not only for sarcomas but among all tumor types ranges between 0.3% and 1.3%, which is lower compared to conventional treatments. A meta-analysis by Wang et al. in 2018 de novo immune responses but dictates the success of immunotherapies as well. Supported by the stiff ECM, CAFs interact with cancer cells to convey metabolic signals required for cancer cell glycolysis, increased catabolic activity and autophagy. Meanwhile, the rapid consumption of available nutrients by cancer cells and secretion of lactate, create regions of hypoxia and high acidity, which further hinder immune cell function. Importantly, T cells found in hypoxic areas of inflamed sarcoma tumors exhibit a profound mitochondrial dysfunction and lack of cytokine production until after checkpoint blockade, suggesting that antigen recognition and infiltration into tumors alone are insufficient for an antitumor response and that the metabolic TME can directly suppress T cells. However, direct targeting of the hypoxia-sensing pathway, HIF, is not immune specific and thus, it may have conflicting effects on the function of CTLs and anti-tumor therapies. Likewise, metabolic pathways with distinct dependencies on cancer and pro-inflammatory immune cells and macrophages must be exploited to achieve a multi-pronged approach for cancer therapy. While “mechanotherapeutics” alone do not possess any antitumor effects, their application lies on targeting the tumor-induced vasculature and ECM that hamper T cell migration and effector function of resident cells, as such allowing efficient priming of TME for an effective antitumor immune response. Thus, their role although complementary is rather crucial for T cell accumulation, promoting antigen presentation and activation of T cells and suppressing the immune tolerance. It is expected that upcoming studies employing TME normalization strategies will be focused on the stratification and selection of patients based on the immune phenotype   Figure  and metaWith respect to future directions, convergence of artificial intelligence and precision medicine offers a breakthrough in biomarker research and disease diagnosis. Indeed, in sarcomas there is not “one-size fits all” molecular target and each subtype needs to be studied independently. This underlines the need for elaborating new minimally invasive techniques to monitor patient progress at a given time like high-throughput molecular profiling. An ongoing clinical trial, MULTISARC (NCT03784014), assesses the feasibility of next generation sequencing exome in metastatic soft tissue sarcoma patients to identify actionable mutations. Under clinical investigation is also the development of conserved transcription signatures spanning sarcoma subtypes including the Complexity Index in Sarcomas (CINSARC), hypoxia associate signature and genomic grade index Supplementary tables.Click here for additional data file.


Sorsby fundus dystrophy (SFD) is a rare autosomal dominant disorder with complete penetrance affecting the macula. This is caused by a mutation in the TIMP-3. This objective narrative review aims to provide an overview of the pathophysiology, current treatment modalities, and future perspectives. A literature search was performed using “PubMed,” “Web of Science,” “Scopus,” “ScienceDirect,” “Google Scholar,” “medRxiv,” and “bioRxiv.” The molecular mechanisms underlying SFD are not completely understood. Novel advancements in cell culture techniques, including induced pluripotent stem cells, may enable more reliable modeling of SFD. These cell culture techniques aim to shed more light on the pathophysiology of SFD, and hopefully, this may lead to the future development of treatment strategies for SFD. Currently, no gene therapy is available. The main treatment is the use of anti-vascular endothelial growth factors (anti-VEGF) to treat secondary choroidal neovascular membrane (CNV), which is a major complication observed in this condition. If CNV is detected and treated promptly, patients with SFD have a good chance of maintaining a functional central vision. Other treatment modalities have been tried but have shown limited benefit, and therefore, have not managed to be more widely accepted. In summary, although there is no definitive cure yet, the use of anti-VEGF treatment for secondary CNV has provided the opportunity to maintain functional vision in individuals with SFD, provided CNV is detected and treated early. Patients with this condition may become symptomatic during their second decade of life, however, the average onset of the disease is usually during the fourth to fifth decade of life.,2 Prior to the era of anti-vascular endothelial growth (anti-VEGF) injections, SFD led to severe bilateral visual reduction and blindness.,2Sorsby fundus dystrophy (SFD) is a rare hereditary macular dystrophy with an autosomal dominant mode of inheritance and full penetrance. It was first described in the literature published in 1949 by Sorsby et alThe aim of this narrative review is to provide a descriptive overview of this inherited genetic disorder.This descriptive review also aims to answer the following questions:A. What is the prevalence of SFD?B. What are the underlying pathophysiological mechanisms of SFD?C. What are the signs and symptoms of the disease?D. What are the current treatment modalities available for SFD and how effective are they?E. What potential future strategies can be developed to treat the disease?A literature search was performed using “PubMed,” “Web of Science,” “Scopus,” “ScienceDirect,” “Google Scholar,” “medRxiv,” and “bioRxiv.” The main keywords for the literature search were “Sorsby Fundus Dystrophy,” “Choroidal Neovascular Membrane (CNV),” “Photodynamic Therapy (PDT),” “Tissue Inhibitor of Metallproteinase-3 (TIMP-3),” “Vascular Endothelial Growth Factor (VEGF),” “Bevacizumab,” “Ranibizumab,” “Triamcinolone,” “ARPE-19 cells,” and “induced pluripotent stem cells (iPSC).” The author attempted to collect data from manuscripts that were mainly published in the past 15 years so that the most recent and up-to-date information on SFD could be incorporated into this manuscript. Nevertheless, published manuscripts >15 years of age were also used, as they contained pertinent information about SFD. The literature search yielded approximately 300 manuscripts. To comply with the maximum number of references, 101 articles were selected.As this is a narrative review, no ethical approval was required. The authors of this study estimated the prevalence of SFD to be 1 in 220,000. Since the condition is inherited in an autosomal dominant manner with complete penetrance, there is no gender predilection. Hence, both men and women can be equally affected by this rare hereditary macular dystrophy.Due to the paucity of SFD cases, no large epidemiological studies exist that calculate the prevalence of the disease in the general population. The only manuscript that attempts to calculate the prevalence is the manuscript by Christensen et al published in 2017.,2 Deposition of drusen within the posterior pole is typically observed during the initial stages of the disease.,5 Reticular drusen and pseudodrusen may also be observed on dilated fundoscopy. In more advanced stages of the disease, formation of a secondary choroidal neovascular membrane (CNV) or geographic atrophy can be observed.,5 Because the primary affected location of the retina is the macula, the main symptoms include distortion, reduced distant and near vision, impaired color vision, and nyctalopia.,4–6 The peripheral retina may be involved during the process of the disease and loss of ambulatory vision may ensue during the 7th decade of life.,4–6 Prior to the era of anti-VEGF injections, SFD led to severe bilateral visual reduction and blindness.,2Patients with this condition may become symptomatic during their second decade of life; however, the average onset of the disease is usually during the fourth to fifth decade of life.,4 This gene is 1 of the 4 genes encoding enzymes that serve as inhibitors of the matrix metalloproteinases (MMP).,7 The cluster of these 4 genes demonstrates a robust tertiary molecular structure induced by the presence of 6 intramolecular disulfide bonds forming between 12 cysteine residues. All 4 genes halt the breakdown of the extracellular matrix due to their N-terminal domain.,8–10 This is of paramount importance for a wide range of physiological processes at a cellular level, including wound healing, angiogenesis, and tissue remodeling. In addition, TIMP-3 seems to be able to inhibit the interaction of VEGF with its receptor VEGF receptor 2 (VEGFR2) by directly binding to the receptor, thus preventing angiogenesis.,11 Nevertheless, it is still unclear whether TIMP-3 gene is involved in the apoptosis of retinal pigment epithelial (RPE) cells and how this can affect SFD. Unlike TIMP-1, TIMP-2, and TIMP-4, which are soluble, TIMP-3 is insoluble.,12,13SFD is attributed to mutations affecting TIMP-3. 18 mutations of the TIMP-3 gene have been implicated in the manifestation of SFD.,6,14–29 In the United Kingdom, most patients with SFD carry the Ser204Cys mutation in exon 5 of the TIMP-3 gene.Based on the author’s literature review and on the manuscript by Christensen et al, Although most mutations are located closer to the C-terminus of the protein, there are mutations affecting the N-terminus as well, and the severity of the disease cannot be easily correlated with the affected amino acid.,22,24,29 Some authors suggest that some additional risk factors may affect the severity of SFD, such as smoking or age-related macular degeneration (AMD) genetic factors.,17,30The presence of different variants in TIMP-3 makes SFD a condition that exhibits heterogeneity in the severity of different clinical phenotypes.,25,27,28 However, mutations not involving the amino acid cysteine have also been described.,17,23 It is also unclear whether TIMP-3 variants retain their ability to inhibit MMP, although most studies advocate this.,11,18,31 It is postulated that loss of the ability to inhibit MMP leads to a more severe phenotype, but some studies dispute this hypothesis.,17,32Most mutations described in the manuscripts referenced above affecting the amino acid cysteine leading to the dimerization of TIMP-3 variants observed with these mutations.,33,34 As mentioned above, TIMP-3 seems to be able to inhibit the interaction of VEGF with its receptor VEGF receptor 2 (VEGFR2) by directly binding to the receptor, thus preventing angiogenesis.,11 It is postulated that this may represent a pivotal step leading to formation of CNV. Nevertheless, patients with SFD do not seem to have an abnormal choroidal structure, as other studies conducted using mouse animal models have shown.,35 Hence, SFD variants may still retain the ability to impede the VEGF signaling cascade to some extent.,35Another controversial topic is whether various TIMP-3 variants preserve the ability to impede the interaction between VEGF and its receptor. The authors described a series of SFD patients from 2 unrelated families, where not only the eyes were affected, but the patients also exhibited pulmonary involvement. Of note, pulmonary involvement has been described in mouse models with TIMP-3 mutations. Meunier et al also posed the question of whether the substitution of a smaller amino acid with a larger amino acid may lead to a more significant impact in the TIMP-3 protein structure, leading to a more severe clinical phenotype of SFD.,36 However, the early onset of disease associated with the Ser179Cys variant  suggests that age of onset is not solely determined by the change in the size of the variant amino acid. Therefore, the hypothesis proposed by Meunier et al could not be confirmed.,36Another interesting point is the observations of the manuscript by Meunier et al. Tsokolas et al described 2 sisters who suffered from SFD that were referred for lung function tests and bone densitometry, which were both normal. Hence, it could be postulated that SFD can be perceived as a syndromic disease with potentially affected extraocular tissues.In addition, another study using mouse models proposed that mutations in TIMP-3 can potentially lead to osteoporosis.In summary, there are still unanswered questions regarding TIMP-3 expression. Solving these mysteries around TIMP-3 can open new pathways in the development of therapeutic strategies for SFD.,39–41 It is also an integral part of Bruch membrane.,42TIMP-3 is expressed primarily in the RPE and choroidal endothelial cells.,43 Other studies have suggested that this is because of increased deposition of TIMP-3 molecules rather than true overexpression of the gene.,44,45 This is supported by using immuno-staining.,40 Despite the fact that TIMP-3 gene is expressed in both RPE and choroidal endothelial cells, the previously mentioned studies postulate that the accumulation of TIMP-3 protein originates most likely from the RPE rather than the choroidal endothelium, making the RPE the primary location of the changes that eventually lead to SFD.Studies have suggested that increasing age may lead to increased deposition of TIMP-3 on the Bruch membrane.,18,32 The subsequent thickening of Bruch membrane eventually affects RPE function. The subsequent disruption of the RPE–retina barrier may result in macular atrophy or CNV formation.,18,32Mutations in TIMP-3 result in the accumulation in the drusen-type of deposits at the level of Bruch membrane observed clinically on fundoscopy in SFD patients.,43,46,47 Therefore, understanding the underlying pathophysiological mechanisms associated with TIMP-3 variants will shed more light into gaining more insight about SFD and other TIMP-3 associated retinopathies as well and will help devise treatment strategies in the future.At this stage, it is imperative to highlight that TIMP-3 protein accumulation is not only observed in SFD but also in other pathological conditions, including retinitis pigmentosa and AMD, where there is abnormal material deposition at the level of Bruch membrane. In the past, different types of tissues were used for this purpose.–52 In recent years, RPE cells are cultured on porous materials.,53Culture of RPE cells has been used for decades to better understand their roles and functions.,54–59 In addition, the use of porous material allows the study of the interactions between the RPE and choroid, hence the study of the blood–retinal barrier.,60,61 Finally, RPE cells interact with the photoreceptors and play a pivotal role in the regeneration of the photoreceptor outer segment during the visual cycle. This interaction can also be using porous materials.,62 These advantages have made RPE cell cultures very useful in modeling conditions, in which there is a breakdown in the normal RPE function and its interactions with the retina, including drusen formation.,63,64 Such ex-vivo studies have provided important advancements in culturing RPE cells and have highlighted the necessity to use RPE cells derived from live patient human tissue that can provide more reliable insight into different aspects of RPE pathophysiology.There are numerous advantages to the use of porous materials for RPE cell cultures. One such advantage is that the cultured cells exhibit polarization of their membranes, which allows the study and quantification of different secreted proteins and membrane receptors. Pluripotent stem cells preserve the ability to differentiate in any type of cell originating from the 3 germ layers. Pluripotent stem cells were first isolated in 1981, and they derived from mouse embryonic stem cells.,65,66 Human embryonic stem cells followed 17 years later.,65,66 Ever since, studies have achieved to generate RPE cells from human embryonic stem cells via direct and spontaneous differentiation.,67–70A very important novel cell culture technique that seems to be quite promising for studying RPE cells is the use of pluripotent stem cells that can differentiate into RPE cells.,68,70 Furthermore, studies suggest that these cells are superior to other cell lines for modeling human RPE cells.,70–73 These attributes have allowed the use of pluripotent stem cell derived RPE cells in the formation of study models of macular dystrophies and in human clinical trials for AMD and Stargardt disease.,70,74RPE cells originating from pluripotent stem cells exhibit morphological features that are identical to those of the typical human RPE cells. This technique allows the propagation of embryonic stem cells with the same characteristics shared by conventional pluripotent cells.,75 Studies have demonstrated the ability to produce RPE cells from iPSCs.,76–80 RPE cells originating from iPSCs seem to be superior to those originating from embryonic stem cells, because they carry the same genetic information in situ (including mutations) with the patient’s somatic cells  from which they are produced.,77Another ground-breaking evolution is the ability to generate pluripotent stem cells from somatic cells, known as iPSCs.,77,81,82 Nevertheless, iPSCs have not been used to model SFD or study various TIMP-3 mutations. In addition, to date, non-RPE cells or non-human cells have been used in various studies to emulate SFD, but these cell types are not reliable for studying the underlying pathophysiological mechanisms associated with SFD and TIMP-3 mutations.,32,83–85 Therefore, exploiting the novel advancements in cell culture techniques can open new pathways for formulating patient-derived RPE cell types that can allow a more reliable and realistic depiction of the various TIMP-3 variants under in vitro conditions. Studies focusing on the TIMP-3 gene and SFD should incorporate the evaluation of TIMP-3 expression, assessment of the role of the RPE in maintaining the RPE–retina barrier, their role in the renewal of the photoreceptor outer segment during the visual cycle and their interaction with the choroidal vascular network. Such studies will allow us to elucidate the molecular mechanisms that may result in CNV formation or geographic atrophy which are the hallmark clinical features of SFD.The above attributes demonstrated by iPSCs have been used to model AMD and other retinal dystrophies, including Best disease and retinitis pigmentosa. Prior to anti-VEGF treatment, vitamin A was administered to treat SFD related nyctalopia.,86 Low doses of Vitamin A were not efficient in the treatment of nyctalopia, whereas higher doses induced liver toxicity.,86 Hence, Vitamin A has not been widely adopted for the treatment of night blindness associated with SFD.To date, there is no definitive treatment for TIMP-3 mutations leading to SFD. Prior to the introduction of anti-VEGF agents in clinical practice, CNV formation used to lead to rapid irreversible loss of central vision and legal blindness.,6,87 However, thermal photocoagulation did not improve visual acuity and aggressive recurrence of CNV was observed as well.,6,87 Therefore, thermal photocoagulation has been deemed obsolete as treatment modality for was PDT verteporfin. PDT was used as a stand-alone therapy or combined with intravitreal corticosteroid injection. Nevertheless, this treatment approach showed very limited benefits, was unpredictable and was eventaully abandoned.,19,88,89CNV formation is the leading cause of central vision loss in patients with SFD. Prior to the introduction of anti-VEGF agents in clinical practice, thermal photocoagulation has been used as an attempt to treat CNV.,24,90–99Five years later, anti-VEGF treatment was introduced in clinical practice, which this revolutionized the treatment of CNV secondary to SFD. There have been quite a few publications that have evaluated the efficacy and safety of anti-VEGF agents in treating CNV secondary to SFD and they have been summarized in a comprehensive systematic review by Baston et al published in 2021. described a mean follow-up period of 54 months for patients treated for SFD. If CNV is recognized and treated early, this systematic review demonstrated that 51% of eyes can preserve an adequate reading and driving visual acuity  of at least 0.5. Furthermore, prompt treatment allowed 67% of eyes to maintain a functional level of vision using reading aids .,24,90–96 If, however, an established disciform scar has ensued, the use of anti-VEGF treatment is of limited benefit. It must also be pointed out that even after prompt treatment, SFD can still progress either due to geographic atrophy or due to macular scar formation.,98 In fact, Kaye et al described a linear decrease in VA of approximately 0.1 Logarithm of Minimum Angle of Resolution units per year until scar formation after a 5-year follow-up period.,98A systematic review by Baston et al,24,90–99 Some of them have also small follow-up periods,,92,96,97 whereas others have longer follow-up periods.,6,98,99 In addition, there is no consensus on the optimum pattern of anti-VEGF injections, that is, whether a treat and extend (T&E) protocol would be preferable to a pro-re-nata (PRN) approach. Kaye et al and Tsokolas et al advocated a T&E approach, whereas Baston et al did not.,5,98 Ideally, prospective studies with larger number of patients that will be divided into the 2 above treatment groups should be conducted. Nevertheless, such an endeavor is quite challenging to come into fruition due to the rarity of SFD patients, which will subsequently raise doubts about the cost-effectiveness of such an endeavor. Despite the lack of consensus in the pattern of injections, all manuscripts highlight the importance of prompt recognition and treatment of CNV secondary to SFD and acknowledge that the introduction of anti-VEGF agents was a game changer in the clinical outcome of CNV secondary to SFD.,24,90–99All the above published manuscripts have significant limitations, namely their retrospective nature and the limited number of patients/eyes treated. The patient originally presented in 2003 with visual acuities of 20/25 and 20/400 right and left eye respectively. PDT was originally tried with no benefit, which confirmed the observations of other published manuscripts.,89,100 The left eye had no further treatment due to permanent foveal structural damage, whereas the right eye was further treated with series of intravitreal injections of triamcinolone in 3 to 4 monthly intervals and retained an excellent acuity. The author postulates that the anti-inflammatory effect of triamcinolone may be able to modify the secretion of VEGF and tumor necrosis factor-α (TNF-α) regulated by TIMP-3 gene. This case report has a long follow-up period, but it has 2 main weaknesses: its retrospective nature and the fact that only 1 eye was treated. Hence, this case report does not provide robust data to advocate the use of intravitreal triamcinolone as a mainstream treatment for CNV secondary to SFD. In addition, intravitreal steroids such as triamcinolone increase the risk of cataract formation and steroid response/glaucoma. These 2 additional risk factors must be considered as they may pose an additional visual burden in patients with SFD, who are already at risk of losing central vision in a relatively young age. Finally, as with anti-VEGF treatment, there is no benefit with steroid treatment when a disciform scar has already formed, as with the patient’s left eye. Hence, larger prospective studies are needed to provide substantial evidence that could make the use of triamcinolone mainstream. However, such an endeavor is quite challenging to come into fruition due to the rarity of patients with SFD, which will subsequently raise doubts about the cost-effectiveness of such an endeavor.Recently, Spaide published a case report of an SFD patient who was followed-up for approximately 16 years and treated with a series of intravitreal injections of triamcinolone. After a series of triamcinolone intravitreal injections, the treatment was switched to bevacizumab, but CNV became active. Then, intravitreal triamcinolone was introduced again, and the patient responded with minimal signs of exudation and hemorrhage. Due to long-term complications associated with intravitreal triamcinolone, the treatment was switched to subcutaneous adalimumab. The total follow-up period after the initiation of adalimumab was 18 months. Throughout this period, CNV in the right eye remained inactive and the final visual acuity in the right eye was 20/20. Since TIMP-3 can regulate the secretion of TNF-α locally, the author suggests that the use of anti-TNF-α agents such as adalimumab may offer a molecularly oriented approach to treatment and warrants further evaluation. Therefore, larger prospective studies are required to elucidate whether adalimumab could be a good alternative treatment for CNV secondary to SFD. However, as mentioned previously, such prospective studies require large numbers of patients and prolonged follow-ups, which is very difficult to achieve given the paucity in numbers of patients with SFD. In addition, biologic agents such as adalimumab have side effects, mainly immunosuppression. In addition, application for special funding is usually required for biologics, and this process can be quite time consuming; hence, patients with SFD will still need prompt treatment with anti-VEGF agents in the interim, until adalimumab is introduced.Spaide also described the use of adalimumab in the same patient described above for the treatment of SFD.In summary, anti-VEGF agents have changed the visual outcomes of CNV secondary to SFD. They remain the cornerstone of treatment of patients with SFD that develop CNV. Intravitreal steroids and biologics have also been reported, but the currently available data are limited to support their wider use in the treatment of CNV associated with SFD.SFD is a rare inherited retinal dystrophy for which no definitive treatment is currently available. There are still many unanswered questions regarding the underlying molecular pathophysiological mechanisms associated with TIMP-3 mutations. Current cell types and animal models used to understand these mechanisms are not fully reliable. With novel advancements in cell culture techniques and the emergence of iPSCs, there is hope that SFD can be modeled more accurately and reliably. This may open new pathways for developing treatment strategies for SFD which can target TIMP-3.Currently, the main aim is to treat complications of SFD, mainly CNV formation. Prior to the introduction of anti-VEGF agents into clinical practice, CNV was untreatable, leading to irreversible foveal damage and subsequent central visual loss. Anti-VEGF treatment has allowed for the preservation of functional vision of individuals with SFD that develop CNV. Patients with SFD must be warned about this significant complication associated with their diagnosis and they must be educated in the regular use of the Amsler grid so that they can detect metamorphopsia promptly and seek immediate help in their local eye casualty service. This will increase the chances of prompt treatment with anti-VEGF agents and subsequent preservation of vision and quality of life. Patients with SFD should also be educated to avoid smoking and follow a diet rich in fruit and vegetables, as these 2 lifestyle habits can slow down the retinal degeneration induced by TIMP-3 mutations.Intravitreal corticosteroids and subcutaneous adalimumab were recently described in 2 case reports as treatment modalities for CNV secondary to SFD. However, supporting data are limited to advocate their wider use, and their efficacy merits further study.Therefore, anti-VEGF agents remain the cornerstone of treatment for CNV secondary to SFD, provided that this complication is recognized and attended to early.Conceptualization: Georgios Tsokolas.Methodology: Georgios Tsokolas.Writing – original draft: Georgios Tsokolas.Writing – review & editing: Georgios Tsokolas.


Vascular remodeling (VR) is a structural and functional change of blood vessels to adapt to the changes of internal and external environment. It is one of the common pathological features of many vascular proliferative diseases. The process of VR is mainly manifested in the changes of vascular wall structure and function, including intimal hyperplasia, thickening or thinning of media, fibrosis of adventitia, etc. These changes are also the pathological basis of aging and various cardiovascular diseases. Mechanical force is the basis of cardiovascular biomechanics, and the newly discovered mechanical sensitive ion channel Piezo1 is widely distributed in the whole cardiovascular system. Studies have confirmed that Piezo1, a mechanically sensitive ion channel, plays an important role in cardiovascular remodeling diseases. This article reviews the molecular mechanism of Piezo1 in atherosclerosis, hypertension and pulmonary hypertension, in order to provide a theoretical basis for the further study of vascular remodeling. Vascular remodeling (VR) is a frontier field in the research of various cardiovascular diseases in recent years. As a dynamic pathological process, it is the change of the structure and function of blood vessels to adapt to the changes in the internal and external environment –3. The f+, Ca2+ into the cells , consisting of a central anchor, three long beams and three blade-like structures . ObserveIt was demonstrated that Piezo1 can sense and transmit a variety of mechanical forces, including cell membrane tension , shear sIt has also been reported that vascular endothelial cells constantly interact with the intra- and extra-luminal extracellular environment and that endothelial cells are endowed with physical stresses, such as shear stresses and tensile forces, which are sensed and transmitted by Piezo1, leading to altered cell behavior with excessive proliferation, migration and aggregation, forming primitive vascular vasculature and further leading to pathological vascular remodeling , 44–46. 2+ with Ca2+ chelators, but was not altered when intracellular Ca2+ was depleted by the application of toxic carotene , indicating that the Yoda1-induced Ca2+ response is likely to be dependent on the inward flow of extracellular Ca2+. In addition, it was found that the administration of certain mechanical stimuli enhanced the kinetic process of Yoda1-induced Piezo1 channels and prolonged the inactivation of transient currents  and the spider venom peptide GsMTx-4 isolated from tarantula toxin  antagonizes the activation of Piezo1 channels by Yoda1 through competing with Yoda1 for the target of action, thereby inhibiting the Yoda1-induced aortic diastole. Meanwhile, it was also found that although TBMS1 did not significantly inhibit phenylephrine-induced aortic ring contraction, it significantly reduced Yoda1-induced vasodilation, suggesting that TBMS1 directly inhibits Piezo1 channel activity or functions through other unknown mechanisms, thus speculating that TBMS1 may act on Piezo1 in vascular smooth muscle cells to partially inhibit vasoconstriction and thus slow down the onset of vascular remodeling . The aboAtherosclerosis (AS) is the most common type of atherosclerosis, which is characterized by the accumulation of lipids and necrotic tissue in the intima of arteries, forming yellow atheromatous plaques , 70. AS It is well known that inflammation is a key factor driving the development and progression of AS, and that disturbed blood flow shear stress can exacerbate the inflammatory response by damaging vascular endothelial cells , 74–76. Proliferation, apoptosis and migration of vascular smooth muscle cells are important pathological changes in diseases such as atherosclerosis and restenosis and are major pathological features of arterial reconstruction due to mechanical stress , 82. Pie2+ influx following Piezo1 activation by Yoda1, which inhibited pathological aortic luminal hypoelasticity and thus slowed down the formation of atherosclerotic plaques  and murine liver endothelial cells (MLEC) has a protective effect against atherosclerosis. It was also demonstrated that the Chinese herbal medicine monomer salvianolic acid B could inhibit Ca plaques .2+ concentration ([Ca2+] cyt) in pulmonary arterial smooth muscle cells (PASMCs) is the main cause of pulmonary vasoconstriction, while an imbalance in intracellular calcium (Ca2+) homeostasis stimulates PASMCs to proliferate and inhibits their apoptosis, leading to pulmonary vascular remodeling. The imbalance in intracellular calcium (Ca2+) homeostasis stimulates PASMCs to proliferate and inhibits their apoptosis, leading to pulmonary vascular remodeling and driving PAH disease progression  in PAECs. The increased Notch ligands in PAECs, as signal-sending cells, then activate Notch receptors in PASMCs, as signal-receiving cells, and result in pulmonary arteriole muscularization and concentric pulmonary vascular remodeling  is a complex, progressive cardiopulmonary disease characterized by non-specific symptoms such as dyspnoea, fatigue, weakness, angina, and syncope. The main features of PAH are increased pulmonary vascular resistance and elevated pulmonary arterial pressure, ultimately leading to right heart failure and death –99. It hgression , 101–105ard flow , 105–1072+] i) caused by mechanical stimulation of PASMCs , Major basic research projects of Natural Science Foundation of Shandong Province (ZR2020ZD16), Youth Natural Science Foundation of Shandong Province (ZR2020QH340), and Traditional Chinese Medicine Development Science and Technology Foundation of Shandong Province (2019-0051).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Intestinal stem cells (ISCs) play an important role in maintaining intestinal homeostasis via promoting a healthy gut barrier. Within the stem cell niche, gut microbiota linking the crosstalk of dietary influence and host response has been identified as a key regulator of ISCs. Emerging insights from recent research reveal that ISC and gut microbiota interplay regulates epithelial self-renewal. This article reviews the recent knowledge on the key role of ISC in their local environment (stem cell niche) associating with gut microbiota and their metabolites as well as the signaling pathways. The current progress of intestinal organoid culture is further summarized. Subsequently, the key challenges and future directions are discussed. Intestinal homeostasis is maintained by a combination of the GI luminal epithelium, gut microbiota, and intestinal stem cells (ISCs). Recent research has focused on the mechanisms of controlling intestinal homeostasis both in health, injury and recovery conditions. Fortunately, recent technological advancements such as intestinal organoids and organ cultures of the gut have allowed us to gain a deeper understanding of this system.As a natural defense system, the intestinal luminal surface is covered by just one layer of epithelium. However, that layer is an effective barrier that guards against pathogens and toxins that may be harmful to the host ISCs in the crypts of the intestines are rare, non-quiescent, undifferentiated cells, which play a crucial role in regulating the homeostasis and regeneration of tissues. Each crypt contains four to six pluripotent stem cells The aim of this review is thus to review the recent knowledge on the key role of ISC in their local environment (stem cell niche) associating with gut microbiota and their metabolites as well as the signaling pathways. The current development of intestinal organoid culture is further summarized. The key challenges and future directions are further discussed.2, is not only the primary site of nutrient absorption and processing, but also the body's largest endocrine organ The GI tract, which has a surface area of approximately 250 cmThe intestinal barrier is composed of two layers Figure : the outIt is common for intestinal homeostasis to be dysregulated when barrier integrity is disrupted. Maintaining gut homeostasis is achieved through a delicate interplay among ECs, gut microbiome, immune system, as well as ISCs et al. pointed out that stem cells that maintain tissue homeostasis were regulated and supported by the surrounding microenvironment, known as the stem cell niche et al. formally introduced the concept of the ISC niche, which includes nearby Paneth cells, neuronal cells, smooth muscle cells and stromal cells, as well as the extracellular matrix together e.g. EPhB and C-Myc) are overexpressed in the embryonic intestinal epithelium, exceptional crypts are formed with the assistance of villi that are perpendicular to the crypt-villi axis ISCs in the crypts are undifferentiated and are involved in tissue homeostasis and regeneration after injury or inflammation et al., the direct progeny of stem cells, known as TA cells (transit-amplifying cells), would unexpectedly dilute any integrated DNA markers used to demonstrate their stemness on remote adult stem cells. A common method of achieving "stemness," therefore, is the long-term storage of DNA labels. When tissue has been severely damaged, stem cells go through mitosis to maintain DNA markers in the tissue. On the contrary, because they are unable to proliferate, DNA labels may continue to be retained by terminally differentiated cells longer than they would in stem cells ISCs replenish themselves by mitosis, dividing into equal stem mobile clones over time. Such division ensures constant stem cell diversification and differentiation. ISCs are required to sustain themselves for an extended period while also provide upward movement to all differentiated types of tissue in question; this is referred to as their "stemness". Stem cells must also be able to provide upward movement to all differentiated mobile phone types of tissue in question. According to Barker The small intestinal epithelium is made up of one type of absorptive cell and four types of secretory cells , which are differentiated from stem cells and generated from the endoderm et al. had previously discovered that Musashi-1 in mice intestinal tissue was an early marker of stem cells and progenitor cells, and is expressed only in damaged tissue (e.g. tumors) et al. discovered that ISCs controlled the stability in the GI environment through tumor-suppressive autophagy Since ISCs allow for the continuous replacement of intestinal ECs, they are critical for maintaining the mucosal barrier, which is a mechanism that protects the intestinal barrier from pathogen invasion and bodily damage in pathological or physiological conditions that activate chronic or acute inflammation. Irritable bowel syndrome is a disorder related to the gut, involving abnormal GI motility, abnormal absorption or secretion, and visceral hypersensitivity in the presence of a stressful environment. Actually, these symptoms may also be associated with a low density of enteroendocrine cells, which is caused by a low density of ISCs Keeping the intestinal lumen stable can be a complex task, which necessitates interaction among the ECs lining the lumen, ISCs, and gut microbiota. The intestinal microbiota comprises a variety of microbial communities, which include viruses, bacteria, and fungi, which is essential for regulating GI function and immune homeostasis et al. found that the abundance of Bacteroides thetaiotaomicron was significantly reduced in obese patients and inversely related to serum glutamate concentrations et al. discovered a decrease in the abundance of Clostridium spp. and Akkermansia muciniphila, whereas the abundance of Ruminococcus, Sutterella and Streptococcus increased in the gut of T2D patients In healthy state, microbiota variability, as reflected in intestinal bacterial diversity and microbial growth rates, is mainly correlated with genetic, environmental and individual differences Throughout the last decade, researchers have discovered that gut bacteria show a complex interplay with ISCs, which plays an important role in gut health. Therefore, this section aims to summarize the recent developments in regulating intestinal microbiota/ derived metabolites-ISC axis Figure . The effMany studies have established a link between the gut microbiota and ISCs, revealing clear communication at the niche level. ISCs are often used as a tool, which is due to its ability to promote a continuous renewal of the intestinal ECs. It has been shown that the intestinal microbiota has an impact on how the intestinal ECs grows and develops. To discover whether early intestinal microbiota contributes to the development of the intestinal tract, Yu and colleagues examined the differentiation and formation of TJs in four different epithelial cell lines  et al. demonstrated that no differences in ISC numbers or cycling activity were observed in the small intestine of germ-free and conventionally raised mice, which suggested that enteric microbiota did not impact ISCs under a normal condition Remarkably, intestinal microbiota exerts varied influences on ISCs during different intestinal states . In a steady-state condition, normal gut microbiome, featuring with balanced beneficial and potential harmful bacteria, helps to maintain gut health. It seems that gut microbiota under healthy condition probably does not give specific advantage to ISCs. SchoenbornIn contrast, accumulated evidence confirms that intestinal microbiota is essential for ISC activity during injury and repair states. The cytosolic bacterial peptidoglycan sensor Nod2 was found highly expressed in ISCs linking microbes to gut epithelial regeneration et al. found that the intestine's relative quantity of beneficial and invading bacteria was directly related with the production and expression of inflammatory cytokines, which interacted to regulate immune function Akkermansia muciniphila) have been shown to be critical for ISC-mediated epithelial growth and intestinal environment stability in a recent study et al. showed that Lactobacillus reuteri induced intestinal epithelial cell proliferation to repair epithelial damage through an increase in R-spondins and reduced intestinal pro-inflammatory cytokine secretion and serum lipopolysaccharides (LPS) concentrations, thereby activating the Wnt/β-catenin pathway  et al. demonstrated that both invasive bacteria and commensal bacteria could affect adult ECs renewal, demonstrating the critical role of ISCs in maintaining the GI environment's stability and host defense. However, the gut microbiota-intestinal epithelium interaction is still largely unknown due to unknown variables and processes. This is potentially due to the fact that a large number of microbes exist in the gut and are involved in complex regulations.Alteration of intestinal microbiota is characterized as either loss of function and/or gain of function. Opportunistic pathogens and their activities could be obtained or overgrow to promote diseases such as infectious diseases, which is termed gain of function dysbiosis. Loss of function dysbiosis is often due to the suppression of beneficial bacteria and their activities, which can be linked to diseases like IBD and obesity. Changes in the composition of the intestinal microbiota can cause abnormal reprogramming of ISCs via a variety of mechanisms including genetic and epigenetic failure, impaired metabolism, ISC stability, and abnormal immune activation et al. demonstrated that Clostridium butyricum inhibited the growth of high-fat diet (HFD)-induced intestinal tumor development in Apcmin/+ mice through modulation of Wnt signaling and alteration of intestinal microbiome Bacillus subtilis not only attenuated the inflammatory response, stimulated the proliferation of ISCs, facilitated the repair of the intestinal barrier, but also restored intestinal flora balance. Ferments of B. subtilis relieved IBD Chrysanthemum morifolium polysaccharides have been shown to be useful in the treatment of ulcerative colitis (UC) by enhancing the establishment of healthy gut bacteria, restoring balance to the intestinal micro-ecological environment, and regenerating the immune system Given the role of gut microbiota and ISCs in gut health, it has been suggested that manipulation of gut microbiome via colonization of specific beneficial bacteria strains or dietary supplementation of prebiotics may be beneficial in certain intestinal diseases. Chen Gut microbiota is capable of biotransforming either host-derived components such as bile acids or dietary substances to generate various metabolites. Several studies have revealed that GMMs had effects on the host's gut environment as well as the immunological balance.Short-chain fatty acids (SCFAs) as the fermentation products of indigestible carbohydrates, primarily comprising acetate, propionate, and butyrate, are the most studied metabolites of gut bacteria The role of SCFAs on ISCs has been intensively investigated. Since the effect of SCFAs on ISC is confounded by multiple factors, such as diet, gut microbiota, and gut physiology, opposing conclusions have been drawn from recent decades of research  et al. found that SCFAs stimulate EC proliferation through the autonomic nervous system in association with dosage of SCFAs, the uptake of intestinal microorganisms and fermentable fibers In vitro experiments have demonstrated that SCFAs promoted mucosal proliferation + ISC pool et al. suggest that microbial production of butyrate, the main source of energy for colon cells, may provide energy for ISCs and promote its value-added et al. demonstrated that SCFAs promoted intestinal gluconeogenesis through a cAMP-dependent mechanism to control glucose metabolism In earlier years, Sakataet al. illustrated that feeding wheat bran significantly reduced the level of butyrate in the colon and inhibited the proliferation of colon cells However, Boffa in vivo studies, the gut microbiota of animals in different houses may be varied in response to dietary fibers or SCFAs. For the in vitro studies, the concentration and culture conditions used may significantly lead to bias in results. In addition, SCFAs have diverse effects on ISCs through not only influencing ISC proliferation but also modulating genetic pathways that might or might not be associated with ISC proliferation. Some researchers have proposed that the distinct metabolic pathways for energy use associating with epigenetics in differentiated and the undifferentiated colonocytes may contribute to the “butyrate paradox” , indole aldehyde (IAld), indole acrylic acid (IA), indole lactic acid, indole ethanol, indole propionic acid, tryptamine, 3-methylindole, Ahr‑/- mice, which was decreased after treatment of IAA. IAA suppressed β-catenin signals through AhR-mediated β-catenin degradation Rnf43 and Znrf3 by blocking Wnt/catenin signaling and limiting its proliferation, and the gut barrier is restored by AhR signals in response to dietary ligands et al. reported that indole-3-carbinol (I3C) inhibited ISC proliferation through activating AhR, which led to the increased expression of Znfr3 and Rnf43 genes for degrading Wnt et al. also showed that I3C could inhibit ISC proliferation Intestinal microbe-derived indole metabolites influence ISCs through acting as ligand of AhR. It is reported that the expression level of β-catenin was unexpectedly high in the cecum of  Peptostreptococcus russellii to mice resulted in increased production of IA which activated AhR in colonic organoids and macrophage and contributed to elevated expression of Ki67 and proliferation of goblet cells in DSS-induced colitic mice Lactobacillus reuteri enhanced mouse intestinal EC proliferation through generating IAld metabolite which activated AhR in lamina propria lymphocytes to upregulate IL22 and promote ISC proliferation The microbial indole metabolites also affect ISCs activity through interaction indirectly with other cells. Wlodarska showed that oral administration ofet al. reported that LCA increased proliferating cells in colonic crypt of mice in vitro and in vivo is not fully understood.Since high levels of diet-induced secondary bile acids (SBAs), such as lithocholic acid (LCA) and deoxycholic acid (DCA), may contribute to the development of colorectal cancer, it can be expected that SBAs have a role in the proliferation of ISCs Lactobacillus plantarum caused alterations in gut epithelial cell growth by promoting physiological amounts of reactive oxygen species (ROS) from Nox enzyme et al. reported that a zero mutation in the Drosophila PGRP-SD gene was related to increased overgrowth of L. plantarum and its metabolite lactate through activation and generation of ROS lactobacilli and induced regeneration of intestinal ECs via WNT3/β-catenin et al. then corroborated the findings of the aforementioned experiments A study by Jones showed that etc. to promote ISCs proliferation Polyamines are a type of compounds that can be either from dietary source or produced by gut microbiota. It was reported that polyamines may serve as energy source or influence intracellular signaling pathways such as the TGF-βRI/Smad3/4, c-fos and c-jun, Dietary variables may have a direct impact on gut homeostasis either directly or indirectly through regulating the composition of the intestinal microbiota and/or microbial metabolites.et al. identified diet-microbial metabolism feedforward loop that showed a remarkable role in modulating ISC proliferation in the stressed gut. They found that dietary raffinose promoted Lactobacillus reuteri growth which increased metabolism of raffinose to fructose for enhancing glycolysis to support ISC proliferation and renewal Frieling and colleagues have found that HFD increased ISCs activity in the Drosophila fruit fly. Specifically, the intestinal microbiota and the JNK signaling pathway in intestinal cells were both required for this action to occur et al. demonstrated that urolithin A, a major microbial metabolite of phenolics from berries and pomegranate fruits, exerted barrier activity by upregulating epithelial TJ proteins in intestinal ECs via the AhR-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in organoid culture Gut microbiota mediated dietary metabolites showed potential effects on ISCs. Singh During development and in adult tissues homeostasis as well, the Notch pathway plays an essential role in determining cell fate, which is a highly conserved mechanism. The Notch system regulates pro-secretory cell signaling by influencing both signaling and receiving cells through ligand-receptor interactions. The Notch signaling receptor is a single transmembrane protein with functional extracellular (NECD), transmembrane (TM) and intracellular (NICD) structural domains. Delta-like and Senate  families are ligands for Notch. First, the Notch receptor is sheared and glycosylated within the Golgi of the recipient cell, followed by S1 cleavage with TM-NICD. Endosomes transfer processed receptors to the plasma membrane, where it binds to the ligand via Deltex regulation and NUMB inhibition. Secondly, TACE  shears NECD away from the TM-NICD structural domain when it binds to the ligand (S2 cleavage). Lastly, NECD-linked ligands form a complex that is dependent on endocytosis and cycling via Mib ubiquitination in signaling cells. In signal-receiving cells, NICD is released from the TM by-secretase (S3 cleavage), resulting in nuclear translocation and binding to the CSL transcription factor complex, allowing transcription of traditional Notch target genes  Figure  99-103.Notch signaling includes Notch receptors, CSL DNA binding proteins, and Notch ligands (DSL proteins), which determines biological cell development by regulating local intercellular interactions and are essential for the maintenance of ISCs Atoh1, with Hes1 and Atoh1 expression being mutually inhibited Hes1 and Atoh1, which is critical for balancing the destiny of cells between uptake and secretion Atoh1 via ubiquitination Gfi1, a transcription factor that is reliant on Atoh1 expression in the intestine, is essential for the differentiation of Atoh1-specific secretory precursors into enteroendocrine and cupped/pan precursors. An investigation has shown that defective Neurog3 suppression at an early stage was required for sub-stasis to occur in Paneth and mucus cell lines, and that sub-stasis resulted in alterations in the cell type ratio in Gif1-deficient mice. Thus, analogous to Hes1/Atoh1, the relative activity of Gfi1/Neurog3 is implicated in the partitioning of cells into secretory lineages Recent research has indicated that Notch's influence on the ISC spectrum was mediated through its action on et al. found that inhibition of Notch-mediated Tuberous Sclerosis Complex 2 (TSC2) helped to promote the differentiation of ECs et al. revealed that dual cortisol-like kinase 1 (Dclk1) may act as ISCs marker and that restricting the Notch signaling pathway decreased ISC survival, implying that the Notch pathway is critical for ISC-mediated saphenous fossa regeneration. Dclk1 expression in saphenous ECs may potentially be used as a measure of ISC survival after injury et al. then proved that Notch signaling modulated the Bmi1/Lgr5+ ISCs ratio, with suppression of Notch signaling decreasing this ratio and vice versa increasing this ratio. This confirms that Notch signaling can induce asymmetric division in response to intestinal inflammation, establishing a direct link between slow and fast-cycle ISCs et al. observed that Ghrelin rehabilitates gut function after intestinal lesions by activating Notch signaling et al. established that Defacre-expressing Paneth Cells were malleable and that the Notch signaling pathway may induce differentiation of Defacre-expressing Paneth Cells into pluripotent stem cells expressing Lgr5+ crypt base columnar and facilitated acute intestinal regeneration following damage ISC self-renewal is maintained and modulated by Notch signaling. Kapuria Recently, the laboratory of Joshua V. Troll demonstrated for the first time that the MyD88-dependent signal induced by the microflora inhibited the Notch signal, thereby promoting the fate of secretory cells. These results link the activity of the microflora with the Notch pathway. In conclusion, Notch signaling is essential for intestinal homeostasis and repair, which when damaged may lead to chronic inflammation and cancer The Wnt pathway is crucial to early development, organogenesis, tissue regeneration and other physiological processes in the animal embryo, which is a highly conserved class of signaling pathway. It's a collection of several downstream signaling pathways that are activated when the ligand protein Wnt binds to membrane protein receptors. The Wnt signaling pathway enables two distinct modes of communication between cells: intercellular (paracrine) communication and autophagic communication (autocrine). The canonical pathway is comprised of Dvl, GSK3, the Wnt family of secretory proteins, APC, Axin, the Frizzled family of transmembrane receptor proteins, β-catenin, and TCF/LEF transcriptional regulators. The planar cell polarity route is involved in cytoskeletal rearrangement and JNK activation; the Wnt/Ca+ pathway is involved in triggering PLC and PKC; additionally, the intracellular pathway is involved in spindle orientation and asymmetric cell division et al. revealed a previously unknown method of β-catenin activation, the MST4-p-catenin signaling axis. A MST4 deficiency led to a reduction of ISCs and slowed colon cancer formation. Secondly, the MST4-p-catenin axis was elevated, which has been associated with a poor outcome in human colorectal cancer. In conclusion, this study broadens avenues for colon cancer targeted treatment Not only is Wnt the main force in ISC proliferation, but its mutagenesis is also a major factor in colon cancer et al. showed that early microbiota regulation of Erdr1 stimulated the Wnt pathway in ECs, boosted Lgr5+ stem cell production, and promotes colonic mucosal repair, which may be utilized to treat cancer and mucosal ulcers Several studies have found that changes in the Wnt pathway and microbial composition regulated the proliferation of intestinal ECs through Myd88 et al. noted that inhibition of proliferation of ISC in patients with cystic fibrosis (CF) by cystic fibrosis transmembrane conductance regulator (Cftr) may promote Wnt/β-catenin signaling, thereby increasing the risk of gut neoplasia et al. proved that RALs are required for effective ISC regeneration downstream of Wnt signaling. ISC function and Lgr5 positivity decreased, resulting in fast crypt mortality and insensitivity to Wnt inhibition, and impaired tissue regeneration et al. reported that YTHDF1, which is distinctly expressed in ISCs, enhanced β-catenin activity by increasing the translation of Wnt signaling. It contributes to the maintenance of ISCs during regeneration and tumorigenesis et al. proved that deletion of Lats1/2 (the core Hippo kinase) inhibited the Wnt pathway, leading to deficiency of ISC, which is based on transcriptional enhanced associate domain (TEAD). Their team recognized that inhibition of TEAD palmitoylation could prevent the above finding et al. showed that Erk1/2 deficiency resulted in activation of the Ras/Raf cascade through activation of Wnt signaling, which then transduced Akt activity to enhance Wnt/β-catenin pathways as well as the mTOR. This was associated with a decrease in mesenchymal Bmp4, which is a Wnt repressor Nevertheless, several protein ligands and cytokines have the ability to regulate the Wnt signaling. Strubberg +, little is known about Lgr5 post-translational regulation. Novellasdemunt et al. indicated that silencing NEDD4 and NEDD4L increased Wnt activation and the amount of ISCs, hence enhancing tumor predisposition and progression To effectively treat chronic intestinal illnesses, it's necessary to completely understand the developmental mechanisms of ISC. However, there's a dispute about the various ISC lineage hierarchy and segregation theories. Anika Böttcher and colleagues demonstrated a new ecological signal Wnt/PCP pathway in the development of ISC In addition, a study found that increased zinc activity maintained gut integrity through activation of Wnt/β-catenin signaling, shedding insight into the efficient preventative approach of ISC-based exogenous zinc preparations BMP is a growth factor of the TGF-β family, and BMP signaling contains a complex formation of different type 1 and type 2 receptors for serine/threonine kinase activity. Ligand recognition can be obtained from both type 1 and type 2 receptors + stromal cells induced by crypt with oncogenic PI3K + ISC proliferation at the base of the crypt while promoting the allocation of secretory cell lines It has been shown that BMPR1A and SMAD4 mutations cause the human polyposis. If BMPR1A is deleted or overexpressed in mice, ectopic crypt formation is stimulated Likewise, other pathways and factors affect intestinal homeostasis and the progression of GI cancers. JAK-STAT signaling has been proven to control the proliferation of ISC and is negatively regulated by Notch et al. postulated that the intestinal VDR protected mice against dysbiosis by modulating the JAK/STAT system. VDR insufficiency is a major risk factor for colon cancer. They identified potential targets for cancer prevention, the VDR, which protected against the effects of dysbiosis by regulating the JAK/STAT system Vitamin D/vitamin D receptor (VDR) inadequacy is a major risk factor for colon cancer. Zhang + ISCs to a Klf6+ wound-healing cell state, and YAP overexpression led to inhibition of tumor formation In addition, YAP1 activity is implicated in the maintenance of ISCs proliferation which acts as an inhibitor of Hippo pathway downstream effectors and/or Wnt signaling, contributing to ISC maintenance and differentiation in transit-amplified cells. The structural integrity of intestinal EC junctions is the primary signal regulating intestinal homeostasis via the Hippo pathway + ISCs Finally, autophagy and its regulatory mechanisms are involved in the homeostasis and repair of the gut by regulating TJs and influencing the metabolism and the proliferative and regenerative capacity of ISCs et al. successfully cultured crypt extracts from the murine small intestine into self-renewing and differentiating organoids in vitro model that more closely resembles the in vivo environment than traditional two-dimensional (2D) cell culture technique. ISCs replicate the self-renewal seen in vivo in the mature gut, resulting in forming a crypt-villi structure, epithelial polarization and functional lumen In 2009, Sato et al. analyzed somatic mutations in the epithelium of UC using organoids et al. showed by organoid that pks+E. coli induced double-stranded DNA breaks and interstrand crosslinks, and that individuals carrying genotoxic pks+E. coli individuals may be at higher risk of developing CRC + stem cells led to recovery of damaged colonic lesions and the repaired colonic structures displays appropriate epithelial barrier function The 3D organoids have many applications in disease modeling, gene function analysis, and regenerative medicine. UC is characterized by chronic inflammation in the colon, and prolonged exposure to an inflammatory environment predisposes to CRC Although organoids have been vastly superior to normal 2D cell cultures, there are still drawbacks, such as a lack of endothelial cells, immune cells and gut microbes, which limits the study of cell-cell and cell-microbe interactions. To better study the interaction of intestinal cells with other types of cells or gut microbe, the recent development of a human intestinal microfluidic organ-on-a-chip model has been engaged to overcome these challenges. Organ chips can integrate different types of cells including ECs, microvascular endothelium, nerve cells, immune cells as well as their interplay with gut microbes As the 3D organoids have enclosed lumens that may limit their value for transport and coculture studies, recently the microinjection technique shows some advantages. For instance, microinjection of fecal microbes into colon organoids demonstrated that the complex microbiota in feces survived and grew in the colon with little change in complexity et al. constructed autologous jejunal mucosal grafts using biomaterials, which showed that this biological scaffold can be efficiently expanded in vitro and has highly similar biochemical properties to those in vivo, becoming a boon for patients with intestinal failure etc.In addition, Meran The digestive tract has an exceptional capacity for homeostasis, as demonstrated by the efficient absorption and secretion of nutrients. As well, it has a strong mucosal barrier that prevents the invasion of pathogenic bacteria and the excessive renewal of epithelial cells and tumor overgrowth, and generates anti-inflammatory components such as IgA and antimicrobial peptides against GI inflammation. Numerous GI and immunometabolic diseases are closely linked to disturbances in gut homeostasis; therefore, intestinal ECs must repair themselves after various pathogenic lesions. The renewal of the intestinal ECs depends on the regulation and support of the ISC and stem cell niches. To maintain dynamic stability, the niche needs to be continuously reshaped and adapted. Notably, the maintenance of ISC activity, which is tightly regulated by several signaling pathways, is influenced by inflammation, gut microbiota, and GMMs.e.g., Bacillus and Lactobacillus) and GMMs  have been identified to directly influence ISC proliferation, renewal and differentiation. Given that the gut microbiota is comprised of a large community of microbes that can be either healthy or pathogenic, it is assumed that the interaction between gut microbes and ISCs is still largely unknown. Thus, a critical future direction for the field of ISCs is to investigate the variables and processes underlying the interaction between the intestinal microbiota and the intestinal epithelium, and more precisely, which signaling pathways or ligand factors are involved, by utilizing finer and more precise molecular markers.Numerous evidence has demonstrated that gut microbiota or their GMMs help maintain gut health during homeostasis and, of particular note, they play a critical role in regulating ISC activity to protect against injury. Specific bacteria (It should be noted that gut microbiota may indirectly impact the stem cell niche via modulating other cells to regulate ISCs. A latest study revealed complex regulation between the gut microbiota, gut neurons, immune cells and ISCs + ISC at the base of the crypt. Defects in autophagy-related genes not only affect the proliferation and differentiation of ISC, but also cause abnormal secretion of antimicrobial peptides by Paneth cells, leading to diseases with impaired intestinal barrier. Other pathways such as YAP, VDR and JAK/STAT also regulate ISC activity. Indeed, in order to understand the role of ISCs in gut homeostasis, it is essential to investigating the tight regulation of ISC maintenance, proliferation, differentiation and stability.In addition, the proper functioning of ISC is dependent on the influence of various signaling pathways such as Wnt, Notch, BMP, autophagy and so on. For example, the Notch pathway plays an influential role in determining cell fate. Wnt/β-catenin activity contributes to ISC differentiation as well as stability, and mutations that aberrantly activate the Wnt pathway may accelerate the proliferation of undifferentiated progenitor cells, ultimately leading to cancer. In contrast, BMP signaling inhibits the proliferation of Lgr5in vitro high-throughput screening and in vivo studies used in disease modeling and drug development, and has potential for regenerative medicine research.Further, most previous studies on ISCs used drosophila or mouse models of the gut. However, in recent years, there has been a growing interest in human intestinal organoids, achieving an increasing number of exciting achievements. The interaction between ISC and other type of cells or gut microbe could be explored. The new development in primary human organoid culture and organ chip will largely facilitate basic research in precision medicine, bridging the gap between traditional


An increase in intracellular calcium concentrations ([Ca2+]i) is a common event in most stress-induced signal transduction pathways. In recent years, significant progress has been made in research related to the early events of stress signaling in plants, particularly in the identification of primary stress sensors. This review highlights current advances that are beginning to elucidate the mechanisms by which abiotic environmental cues are sensed via Ca2+ signals. Additionally, this review discusses important questions about the integration of the sensing of multiple stress conditions and subsequent signaling responses that need to be addressed in the future.Plants are exposed to various environmental stresses. The sensing of environmental cues and the transduction of stress signals into intracellular signaling are initial events in the cellular signaling network. As a second messenger, Ca Plants encounter various abiotic stresses in their natural environment, for example, mechanical stimuli, drought, flood, cold, and salinity stress. These adverse conditions often limit plant growth and crop productivity worldwide . To survTypically, a primary abiotic stress sensor is required to perceive external environmental changes; in response to such changes, the sensor remodels signal transduction pathways and initiates appropriate responses that allow the plant to adapt to the stress condition. Thus, perception of the external environment by a sensor is the earliest step in the conversion of external stimuli into cellular signals by plants.2+), an indispensable second messenger, is considered to be a critical element by which plants modulate a complicated network of signaling pathways to respond to various abiotic stresses (2+]i) is one of the earliest signaling events when plants are challenged with abiotic stimuli, and the resulting Ca2+ signaling regulates many processes in plants, including transcriptional regulation and subsequent physiological as well as developmental responses , the link between abiotic stress sensors and the Ca2+ influx channels that are regulated by these sensors is still largely unknown. Here, we mainly discuss recent findings related to Ca2+ signaling to reveal the mechanisms by which plants sense stressful environment; these findings have advanced our understanding of plant sensory mechanisms.In the resting state, i increase by activating certain Ca2+-permeable channels, allowing plant cells to perceive changes in the physical properties of the membrane -like proteins (MSLs), Mid1-complement activity protein channels (MCAs), two-pore potassium channels (TPKs), Piezo channels (PZO), and reduced hyperosmolality induced i and is hypersensitive to growth inhibition by Na+  to form glycosylinositol phosphorylceramide (GIPC) sphingolipids in the external layer of the plasma membrane, and it is essential for NaCl-triggered depolarization of the cell-surface potential  mutant, indicating that TPC1 mediates Ca2+ release from the vacuole and facilitates the propagation of Ca2+ waves  is required for NaCl-activated Ca2+ influx through the plasma membrane in root epidermal cells. The loss-of-function mutation of AtANN4 also disrupts the salt-induced [Ca2+]i increases in Arabidopsis , which was the first potential cold sensor to be identified in rice. Overexpression of COLD1 significantly improves survival rates after chilling treatment, whereas mutants with deficiency or decreased expression of COLD1 are sensitive to chilling stress , which is produced extracellularly in response to external stresses and internal cues. Extracellular hydrogen peroxide (eH2O2) plays a vital role in many physiological processes during the lifecycle of plants, such as root development, pollen tube growth, organ wilting, and responses to biotic and abiotic stress (Reactive oxygen species (ROS) play a key role in plant cell signaling. Plant cells generate various ROS, including hydrogen peroxide , which regulates ABA signaling and stomatal closure in guard cells  and stimulating stomatal closure  EF-hands and promotes H2O2 which then subsequently activates Ca2+ channels in response to a range of abiotic stresses  mutants in Arabidopsis and showed that HPCA1 encodes a leucine-rich repeat receptor kinase that localizes to the cell surface. It was the first molecular component found to be responsible for sensing eH2O2  and Shenzhen Peacock Plan (KQTD2017032715165926).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.