Inflammation- and Gut-Homing Macrophages,Engineered to De Novo Overexpress Active Vitamin D,Promoted the Regenerative Function of Intestinal Stem Cells

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gut. Available drugs aim to suppress gut inflammation. These drugs have significantly delayed disease progression and improved patients’ quality of life. However, the disease continues to progress, underscoring the need to develop novel therapies. Aside from chronic gut inflammation, IBD patients also experience a leaky gut problem due to damage to the intestinal epithelial layer. In this regard, epithelial regeneration and repair are mediated by intestinal stem cells. However, no therapies are available to directly enhance the intestinal stem cells’ regenerative and repair function. Recently, it was shown that active vitamin D, i.e., 1,25-dihydroxyvitamin D or 1,25(OH)₂D, was necessary to maintain Lgr5⁺ intestinal stem cells, actively cycling under physiological conditions. In this study, we used two strategies to investigate the role of 1,25(OH)₂D in intestinal stem cells’ regenerative function. First, to avoid the side effects of systemic high 1,25(OH)₂D conditions, we used our recently developed novel strategy to deliver locally high 1,25(OH)₂D concentrations specifically to inflamed intestines. Second, because of the Lgr5⁺ intestinal stem cells’ active cycling status, we used a pulse-and-chase strategy via 5-bromo-2′-deoxyuridine (BrdU) labeling to trace the Lgr5⁺ stem cells through the whole epithelial regeneration process. Our data showed that locally high 1,25(OH)₂D concentrations enhanced intestinal stem cell migration. Additionally, the migrated cells differentiated into mature epithelial cells. Our data, therefore, suggest that local delivery of high 1,25(OH)₂D concentrations is a promising strategy to augment intestinal epithelial repair in IBD patients.     

Metformin Protects the Intestinal Barrier by Activating Goblet Cell Maturation and Epithelial Proliferation in Radiation-Induced Enteropathy

Radiotherapy or accidental exposure to high-dose radiation can cause severe damage to healthy organs. The gastrointestinal (GI) tract is a radiation-sensitive organ of the body. The intestinal barrier is the first line of defense in the GI tract, and consists of mucus secreted by goblet cells and a monolayer of epithelium. Intestinal stem cells (ISCs) help in barrier maintenance and intestinal function after injury by regulating efficient regeneration of the epithelium. The Wnt/β-catenin pathway plays a critical role in maintaining the intestinal epithelium and regulates ISC self-renewal. Metformin is the most widely used antidiabetic drug in clinical practice, and its anti-inflammatory, antioxidative, and antiapoptotic effects have also been widely studied. In this study, we investigated whether metformin alleviated radiation-induced enteropathy by focusing on its role in protecting the epithelial barrier. We found that metformin alleviated radiation-induced enteropathy, with increased villi length and crypt numbers, and restored the intestinal barrier function in the irradiated intestine. In a radiation-induced enteropathy mouse model, metformin treatment increased tight-junction expression in the epithelium and inhibited bacterial translocation to mesenteric lymph nodes. Metformin increased the number of ISCs from radiation toxicity and enhanced epithelial repair by activating Wnt/β-catenin signaling. These data suggested that metformin may be a potential therapeutic agent for radiation-induced enteropathy.     

Modeling Intestinal Stem Cell Function with Organoids

Intestinal epithelial cells (IECs) are crucial for the digestive process and nutrient absorption. The intestinal epithelium is composed of the different cell types of the small intestine (mainly, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, and tuft cells). The small intestine is characterized by the presence of crypt-villus units that are in a state of homeostatic cell turnover. Organoid technology enables an efficient expansion of intestinal epithelial tissue in vitro. Thus, organoids hold great promise for use in medical research and in the development of new treatments. At present, the cholinergic system involved in IECs and intestinal stem cells (ISCs) are attracting a great deal of attention. Thus, understanding the biological processes triggered by epithelial cholinergic activation by acetylcholine (ACh), which is produced and released from neuronal and/or non-neuronal tissue, is of key importance. Cholinergic signaling via ACh receptors plays a pivotal role in IEC growth and differentiation. Here, we discuss current views on neuronal innervation and non-neuronal control of the small intestinal crypts and their impact on ISC proliferation, differentiation, and maintenance. Since technology using intestinal organoid culture systems is advancing, we also outline an organoid-based organ replacement approach for intestinal diseases.     

Intestinal Radiation Protection and Mitigation by Second-Generation Probiotic Lactobacillus-reuteri Engineered to Deliver Interleukin-22

(1) Background: The systemic administration of therapeutic agents to the intestine including cytokines, such as Interleukin-22 (IL-22), is compromised by damage to the microvasculature 24 hrs after total body irradiation (TBI). At that time, there is significant death of intestinal microvascular endothelial cells and destruction of the lamina propria, which limits drug delivery through the circulation, thus reducing the capacity of therapeutics to stabilize the numbers of Lgr5+ intestinal crypt stem cells and their progeny, and improve survival. By its direct action on intestinal stem cells and their villus regeneration capacity, IL-22 is both an ionizing irradiation protector and mitigator. (2) Methods: To improve delivery of IL-22 to the irradiated intestine, we gavaged Lactobacillus-reuteri as a platform for the second-generation probiotic Lactobacillus-reuteri-Interleukin-22 (LR-IL-22). (3) Results: There was effective radiation mitigation by gavage of LR-IL-22 at 24 h after intestinal irradiation. Multiple biomarkers of radiation damage to the intestine, immune system and bone marrow were improved by LR-IL-22 compared to the gavage of control LR or intraperitoneal injection of IL-22 protein. (4) Conclusions: Oral administration of LR-IL-22 is an effective protector and mitigator of intestinal irradiation damage.     

Lingual Epithelial Stem Cells and Organoid Culture of Them

As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.     

NOD-Like Receptors in Intestinal Homeostasis and Epithelial Tissue Repair

The intestinal epithelium constitutes a dynamic physical barrier segregating the luminal content from the underlying mucosal tissue. Following injury, the epithelial integrity is restored by rapid migration of intestinal epithelial cells (IECs) across the denuded area in a process known as wound healing. Hence, through a sequence of events involving restitution, proliferation and differentiation of IECs the gap is resealed and homeostasis reestablished. Relapsing damage followed by healing of the inflamed mucosa is a hallmark of several intestinal disorders including inflammatory bowel diseases (IBD). While several regulatory peptides, growth factors and cytokines stimulate restitution of the epithelial layer after injury, recent evidence in the field underscores the contribution of innate immunity in controlling this process. In particular, nucleotide-binding and oligomerization domain-like receptors (NLRs) play critical roles in sensing the commensal microbiota, maintaining homeostasis, and regulating intestinal inflammation. Here, we review the process of intestinal epithelial tissue repair and we specifically focus on the impact of NLR-mediated signaling mechanisms involved in governing epithelial wound healing during disease.     

Corneal Epithelial Regeneration: Old and New Perspectives

Corneal blindness is the fifth leading cause of blindness worldwide, and therapeutic options are still often limited to corneal transplantation. The corneal epithelium has a strong barrier function, and regeneration is highly dependent on limbal stem cell proliferation and basement membrane remodeling. As a result of the lack of corneal donor tissues, regenerative medicine for corneal diseases affecting the epithelium is an area with quite advanced basic and clinical research. Surgery still plays a prominent role in the treatment of epithelial diseases; indeed, innovative surgical techniques have been developed to transplant corneal and non-corneal stem cells onto diseased corneas for epithelial regeneration applications. The main goal of applying regenerative medicine to clinical practice is to restore function by providing viable cells based on the use of a novel therapeutic approach to generate biological substitutes and improve tissue functions. Interest in corneal epithelium rehabilitation medicine is rapidly growing, given the exposure of the corneal outer layers to external insults. Here, we performed a review of basic, clinical and surgical research reports on regenerative medicine for corneal epithelial disorders, classifying therapeutic approaches according to their macro- or microscopic target, i.e., into cellular or subcellular therapies, respectively.     

The Increase of miR-195-5p Reduces Intestinal Permeability in Ulcerative Colitis,Modulating Tight Junctions’ Expression

Defects in the intestinal epithelial barrier functions characterize inflammatory conditions such as Inflammatory Bowel Disease (IBD). Overexpression of pro-inflammatory cytokines such as TNF-α, IL-1B, IL-6 and INF-γ trigger epithelial damage. These cytokines are due to upregulation of claudin-2 (CLDN2) that form a pore channel, resulting in redistribution of TJs and an alteration of barrier permeability. Recently, we demonstrated that miR-195-5p is able to regulate CLDN2 and indirectly also CLDN1 in intestinal epithelial cells. Now, we aimed to investigate the modulation of miR-195-5p on the expression of CLDN2 and other TJs under inflammatory conditions induced by TNF-α. We demonstrated that miR-195-5p also modulated the expression of CLDN2 levels after stimulation with TNF-α. In addition, we discovered the role of miR-195-5p in the integrity of the intestinal barrier and in promoting the restoration of the intestinal epithelial. Moreover, we established that replacement of miR-195-5p attenuated the colonic inflammatory response in DSS-induced, colitis and it reduced colonic permeability. In conclusion, our data revealed the role of miR-195-5p in intestinal inflammation in ulcerative colitis, suggesting a potential pharmacological target for new therapeutic approaches.     

Self-Renewal and Cancers of the Gastric Epithelium: An Update and the Role of the Lectin TFF1 as an Antral Tumor Suppressor

In 2020, gastric cancer was the fourth leading cause of cancer deaths globally. About 90% of gastric cancers are sporadic and the vast majority are correlated with Helicobacter pylori infection; whereas familial clustering is observed in about 10% of cases. Gastric cancer is now considered to be a disease originating from dysregulated self-renewal of the gastric glands in the setting of an inflammatory environment. The human stomach contains two types of gastric units, which show bi-directional self-renewal from a complex variety of stem cells. This review focuses on recent progress concerning the characterization of the different stem cell populations and the mainly mesenchymal signals triggering their stepwise differentiation as well as the genesis of pre-cancerous lesions and carcinogenesis. Furthermore, a model is presented (Lectin-triggered Receptor Blocking Hypothesis) explaining the role of the lectin TFF1 as an antral tumor suppressor possibly regulating Lgr5⁺ antral stem cells in a paracrine or maybe autocrine fashion, with neighboring antral gland cells having a role as niche cells.     

Synchrotron Infrared Microspectroscopy for Stem Cell Research

Stem cells have shown great potential functions for tissue regeneration and repair because of their unlimited self-renewal and differentiation. Stem cells reside in their niches, making them a hotspot for the development and diagnosis of diseases. Complex interactions between niches and stem cells create the balance between differentiation, self-renewal, maturation, and proliferation. However, the multi-facet applications of stem cells have been challenged since the complicated responses of stem cells to biological processes were explored along with the limitations of current systems or methods. Emerging evidence highlights that synchrotron infrared microspectroscopy, known as synchrotron radiation-based Fourier transform infrared microspectroscopy, has been investigated as a potentially attractive technology with its non-invasive and non-biological probes in stem cell research. With their unique vibration bands, the quantitative mapping of the content and distribution of biomolecules can be detected and characterized in cells or tissues. In this review, we focus on the potential applications of synchrotron infrared microspectroscopy for investigating the differentiation and fate determination of stem cells.     

Epidermal Stem Cells and Their Epigenetic Regulation

Stem cells play an essential role in embryonic development, cell differentiation and tissue regeneration. Tissue homeostasis in adults is maintained by adult stem cells resident in the niches of different tissues. As one kind of adult stem cell, epidermal stem cells have the potential to generate diversified types of progeny cells in the skin. Although its biology is still largely unclarified, epidermal stem cells are widely used in stem cell research and regenerative medicine given its easy accessibility and pluripotency. Despite the same genome, cells within an organism have different fates due to the epigenetic regulation of gene expression. In this review, we will briefly discuss the current understanding of epigenetic modulation in epidermal stem cells.     

Toward Xeno-Free Differentiation of Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells

The small intestinal epithelium has an important role in nutrition, but also in drug absorption and metabolism. There are a few two-dimensional (2D) patient-derived induced pluripotent stem cell (iPSC)-based intestinal models enabling easy evaluation of transcellular transport. It is known that animal-derived components induce variation in the experimental outcomes. Therefore, we aimed to refine the differentiation protocol by using animal-free components. More specifically, we compared maturation of 2D-cultured iPCSs toward small intestinal epithelial cells when cultured either with or without serum, and either on Geltrex or on animal-free, recombinant laminin-based substrata. Differentiation status was characterized by qPCR, immunofluorescence imaging, and functionality assays. Our data suggest that differentiation toward definitive endoderm is more efficient without serum. Both collagen- and recombinant laminin-based coating supported differentiation of definitive endoderm, posterior definitive endoderm, and small intestinal epithelial cells from iPS-cells equally well. Small intestinal epithelial cells differentiated on recombinant laminin exhibited slightly more enterocyte specific cellular functionality than cells differentiated on Geltrex. Our data suggest that functional small intestinal epithelial cells can be generated from iPSCs in serum-free method on xeno-free substrata. This method is easily converted to an entirely xeno-free method.     

Fractone Stem Cell Niche Components Provide Intuitive Clues in the Design of New Therapeutic Procedures/Biomatrices for Neural Repair

The aim of this study was to illustrate recent developments in neural repair utilizing hyaluronan as a carrier of olfactory bulb stem cells and in new bioscaffolds to promote neural repair. Hyaluronan interacts with brain hyalectan proteoglycans in protective structures around neurons in perineuronal nets, which also have roles in the synaptic plasticity and development of neuronal cognitive properties. Specialist stem cell niches termed fractones located in the sub-ventricular and sub-granular regions of the dentate gyrus of the hippocampus migrate to the olfactory bulb, which acts as a reserve of neuroprogenitor cells in the adult brain. The extracellular matrix associated with the fractone stem cell niche contains hyaluronan, perlecan and laminin α5, which regulate the quiescent recycling of stem cells and also provide a means of escaping to undergo the proliferation and differentiation to a pluripotent migratory progenitor cell type that can participate in repair processes in neural tissues. Significant improvement in the repair of spinal cord injury and brain trauma has been reported using this approach. FGF-2 sequestered by perlecan in the neuroprogenitor niche environment aids in these processes. Therapeutic procedures have been developed using olfactory ensheathing stem cells and hyaluronan as a carrier to promote neural repair processes. Now that recombinant perlecan domain I and domain V are available, strategies may also be expected in the near future using these to further promote neural repair strategies.     

Baicalin-Induced Autophagy Preserved LPS-Stimulated Intestinal Cells from Inflammation and Alterations of Paracellular Permeability

Several studies have demonstrated a relevant role of intestinal epithelial cells in the immune response and in chronic inflammatory conditions, including ulcers, colitis, and Crohn’s disease. Baicalin (BA), extracted from the root of Scutellaria baicalensis, has various beneficial healthy effects, including anti-inflammatory activity. However, few studies have evaluated BA effects on autophagic signaling in epithelial cell response to inflammatory stimuli. To explore possible beneficial effects of BA, HT-29 cells were exposed to lipopolysaccharide (LPS), in presence or absence of BA, for 4 h. We evaluated mRNA levels of autophagy-related genes and cytokines, triggering inflammatory response. Furthermore, the expression of claudin 1, involved in the regulation of paracellular permeability was analyzed. BA treatment repressed LPS-induced expression of TNF-α and IL-1β. The down-regulation of autophagy-related genes induced by LPS was counteracted by cell pretreatment with BA. Under these conditions, BA reduced the NF-κB activation caused by LPS. Also, BA restored mRNA and protein levels of claudin 1, which were reduced by LPS. In conclusion, in intestinal epithelial cells BA regulates the NF-κB activation and modulates both autophagic and inflammatory processes, leading to an improvement of paracellular permeability. These results suggest that the anti-inflammatory effects of BA can be associated to the regulation of autophagic flux.     

Superoxide Dismutase 3-Transduced Mesenchymal Stem Cells Preserve Epithelial Tight Junction Barrier in Murine Colitis and Attenuate Inflammatory Damage in Epithelial Organoids

Superoxide dismutase 3 (SOD3), also known as extracellular superoxide dismutase, is an enzyme that scavenges reactive oxygen species (ROS). It has been reported that SOD3 exerts anti-inflammatory abilities in several immune disorders. However, the effect of SOD3 and the underlying mechanism in inflammatory bowel disease (IBD) have not been uncovered. Therefore, in the present study, we investigated whether SOD3 can protect intestinal cells or organoids from inflammation-mediated epithelial damage. Cells or mice were treated with SOD3 protein or SOD3-transduced mesenchymal stem cells (MSCs). Caco-2 cells or intestinal organoids stimulated with pro-inflammatory cytokines were used to evaluate the protective effect of SOD3 on epithelial junctional integrity. Dextran sulfate sodium (DSS)-induced colitis mice received SOD3 or SOD3-transduced MSCs (SOD3-MSCs), and were assessed for severity of disease and junctional protein expression. The activation of the mitogen-activated protein kinase (MAPK) pathway and elevated expression of cytokine-encoding genes decreased in TNF-α-treated Caco-2 cells or DSS-induced colitis mice when treated with SOD3 or SOD3-MSCs. Moreover, the SOD3 supply preserved the expression of tight junction (ZO-1, occludin) or adherence junction (E-cadherin) proteins when inflammation was induced. SOD3 also exerted a protective effect against cytokine- or ROS-mediated damage to intestinal organoids. These results indicate that SOD3 can effectively alleviate enteritis symptoms by maintaining the integrity of epithelial junctions and regulating inflammatory- and oxidative stress.     

Distinct Expression Patterns of Cxcl12 in Mesenchymal Stem Cell Niches of Intact and Injured Rodent Teeth

Specific stem cell populations within dental mesenchymal tissues guarantee tooth homeostasis and regeneration throughout life. The decision between renewal and differentiation of stem cells is greatly influenced by interactions with stromal cells and extracellular matrix molecules that form the tissue specific stem cell niches. The Cxcl12 chemokine is a general marker of stromal cells and plays fundamental roles in the maintenance, mobilization and migration of stem cells. The aim of this study was to exploit Cxcl12-GFP transgenic mice to study the expression patterns of Cxcl12 in putative dental niches of intact and injured teeth. We showed that endothelial and stromal cells expressed Cxcl12 in the dental pulp tissue of both intact molars and incisors. Isolated non-endothelial Cxcl12⁺ dental pulp cells cultured in different conditions in vitro exhibited expression of both adipogenic and osteogenic markers, thus suggesting that these cells possess multipotent fates. Taken together, our results show that Cxcl12 is widely expressed in intact and injured teeth and highlight its importance as a key component of the various dental mesenchymal stem cell niches.     

Regeneration of Articular Cartilage in Lizard Knee from Resident Stem/Progenitor Cells

The epiphysis of femur and tibia in the lizard Podarcis muralis can extensively regenerate after injury. The process involves the articular cartilage and metaphyseal (growth) plate after damage. The secondary ossification center present between the articular cartilage and the growth plate is replaced by cartilaginous epiphyses after about one month of regeneration at high temperature. The present study analyzes the origin of the chondrogenic cells from putative stem cells located in the growing centers of the epiphyses. The study is carried out using immunocytochemistry for the detection of 5BrdU-labeled long retaining cells and for the localization of telomerase, an enzyme that indicates stemness. The observations show that putative stem cells retaining 5BrdU and positive for telomerase are present in the superficial articular cartilage and metaphyseal growth plate located in the epiphyses. This observation suggests that these areas represent stem cell niches lasting for most of the lifetime of lizards. In healthy long bones of adult lizards, the addition of new chondrocytes from the stem cells population in the articular cartilage and the metaphyseal growth plate likely allows for slow, continuous longitudinal growth. When the knee is injured in the adult lizard, new populations of chondrocytes actively producing chondroitin sulfate proteoglycan are derived from these stem cells to allow for the formation of completely new cartilaginous epiphyses, possibly anticipating the re-formation of secondary centers in later stages. The study suggests that in this lizard species, the regenerative ability of the epiphyses is a pre-adaptation to the regeneration of the articular cartilage.