Epithelial Stem Cell Differentiation Research Articles

Role of Immunohistochemistry in the Diagnosis of Pilomatrical Tumors

Pilomatrical skin tumors harbor mutations in CTNNB1, which encodes for β-catenin, a downstream effector of the Wnt signaling pathway responsible for the differentiation, proliferation, and adhesion of epithelial stem cells. Therefore, downstream molecules, such as CDX2, LEF-1, and SATB2, in the Wnt signaling pathway could be useful diagnostic markers. Here, we sought to investigate the potential of immunohistochemistry (IHC) to differentiate between pilomatricoma and pilomatrical carcinoma, as well as from other cutaneous adnexal tumors. We studied 88 cases of cutaneous tumors (14 pilomatrical carcinomas, 18 pilomatricomas, 13 basal cell carcinomas, 12 squamous cell carcinomas, 12 sebaceous carcinomas, 10 Merkel cell carcinomas, 7 trichoblastomas, and 2 hidradenocarcinomas) using a broad panel of IHC markers: β-catenin, SATB2, CDX2, LEF1, Ber-EP4, and PRAME. Pilomatricoma and pilomatrical carcinoma displayed >75% nuclear staining for β-catenin. CDX2 also strongly stained pilomatrical tumors; however, the staining distribution was limited in pilomatricoma and more widespread in pilomatrical carcinoma. But, overall, it was less than β-catenin. SATB2 and Ber-EP4 expressions were noted only in a subset of both pilomatrical carcinoma and pilomatricoma, whereas LEF-1 showed strong, diffuse nuclear positivity in both pilomatricoma and pilomatrical carcinoma. Among the IHC markers evaluated, none could distinguish between pilomatricoma and pilomatrical carcinoma. However, the combined use of β-catenin with CDX2 markers may assist in not only confirming the pilomatrical nature of the proliferation but also in differentiating benign from malignant cases when there is a significant presence of CDX2 staining. Despite these findings, the diagnosis should continue to primarily depend on a thorough histopathologic examination.

In vitro differentiation of human amniotic epithelial stem cells into keratinocytes regulated by OPN3.

Human amniotic epithelial stem cells (hAESCs) are regarded as potential alternatives to keratinocytes (KCs) used for skin wound healing. Light is an alternative approach for inducing stem cell differentiation. Opsins (OPNs), a family of light-sensitive, G protein-coupled receptors, play a multitude of light-dependent and light-independent functions in extraocular tissues. However, it remains unclear whether the light sensitivity and function of OPNs are involved in light-induced differentiation of hAESCs to KCs. Herein, we determine the role of OPNs in differentiation of hAESCs into KCs through cell and molecular biology approaches invitro. It is shown that mRNA expression of OPN3 in the amniotic membrane and hAESCs was higher than the other four primary OPNs by RT-qPCR analysis. Changes in OPN3 gene expression had a significant impact on cell proliferation, stemness and differentiation capability of hAESCs. Furthermore, we found a significant upregulation of OPN3, KRT5 and KRT14 with hAESCs treated at 3 × 33 J/cm2 irradiation from blue-light LED. Taken together, these results suggest that OPN3 acts as a positive regulator of differentiation of hAESCs into KCs. This study provides a novel insight into photosensitive OPNs associated with photobiomodulation(PBM)-induced differentiation in stem cells.

NRG1 Regulates Proliferation, Migration and Differentiation of Human Limbal Epithelial Stem Cells.

Limbal epithelial stem/progenitor cells (LESCs) proliferate, migrate and differentiate into mature corneal epithelium cells (CECs) that cover the ocular surface. LESCs play a crucial role in the maintenance and regeneration of the corneal epithelium, and their dysfunction can lead to various corneal diseases. Neuregulin 1 (NRG1) is a member of the epidermal growth factor family that regulates the growth and differentiation of epithelial tissues. Here, we depicted the dynamic transcriptomic profiles during human CEC differentiation, identifying six gene co-expression modules that were specific to different differentiation stages. We found that the expression of NRG1 was high in human LESCs and decreased dramatically upon differentiation. Knockdown of NRG1 significantly inhibited LESC proliferation and upregulated the expression of the terminal differentiation marker genes KRT3, KRT12 and CLU. In addition, the scratch wound closure assay showed that knockdown of NRG1 attenuated wound closure of LESCs over 24 h. Together, we dissected the transcriptional regulatory dynamics during CEC differentiation and identified NRG1 as a key regulator that promoted LESC proliferation and migration and maintained the undifferentiated state.

Mettl14-mediated m6 A modification regulates the abnormal differentiation of small intestinal epithelial stem cells in diabetic state.

Diabetes mellitus (DM) and its related complications are a global epidemic characterized by high morbidity and mortality. However, little is known about diabetic enteropathy (DE) and its the potential underlying mechanism. Intestinal epithelial stem cells (IESCs) were harvested from experimental mice, and the levels of dominant N6-methyladenosine (m6 A)-related enzyme were detected by RT-PCR, Western blotting, immunohistochemistry. The role of Mettl14 in the abnormal differentiation of intestinal epithelial cells (IECs) during DM was confirmed by knockdown experiments. RT-PCR, MeRIP, and bioinformatics analysis were carried out to confirm the downstream target of Mettl14. Through bioinformatics analysis, RT-PCR, and Western blotting, we further analyzed the differentiation-related gene in the IECs from mice with DM. In this study, the levels of Mettl14 and m6 A were higher in db/db mice than that in control mice. And abnormal differentiation of IECs in DM was associated with Mettl14 overexpression. Additionally, Mettl14 is a major determinant of IESCs identity and organoid-forming upon DM state. Mechanistically, we revealed that the candidate binding target of Mettl14 was Fzd2 mRNA and affected Fzd2 stability. Moreover, Mettl14 downregulation was observed to attenuate the abnormal differentiation of IECs through modulating Fzd2 m6A modification in DM state. Together, our results provide definitive evidence for the essential role of Mettl14 in differentiation of IESCs in DM state.

Establishment of a vaginal organoid system to study local resident memory T cell differentiation

Abstract Over a million sexually transmitted diseases (STDs) are acquired every day worldwide, many of which preferentially target the female reproductive tract (FRT). Resident memory T cells (TRM) in the vaginal mucosa have proven protective roles against these STDs. Accordingly, a crucial objective of STD vaccinations is to establish a higher quantity and quality of TRM in the FRT. The vaginal epithelium has been implicated in regulation of local TRM differentiation and function, but the biological nature of this interaction has not been investigated. Here, we established a murine three dimensional (3D) vaginal organoid-CD8 coculture system to examine epithelial-T cell interactions at the molecular level. Differentiation of single epithelial stem cells led to the formation of 3D epithelial organoids. These organoids can be maintained long-term through serial passage and structurally resembled vaginal epithelium with a multilayer specialized epithelium. After culturing activated CD8 T cells with the organoids, we assessed the transcriptional and phenotypic composition of the T cells. We found that cocultured CD8 T cells were integrated into the extracellular matrix and acquired phenotypic and functional traits of in vivo TRM. This TRM-biased differentiation could be suppressed by a small molecule inhibitor of the cytokine TGF-β. Overall, the vaginal organoid coculture model recapitulates the in vivo environment far better than traditional 2D cell culture, while retaining high-throughput abilities. Importantly, it will enable highly reductionist experiments to examine the complex epithelial cell-T cell crosstalk to devise targeted approaches for improving CD8 T cell mediated immunosurveillance in the FRT. Supported by grants from NIH (2P20GM109035 & P20GM121298) and by the Searle Scholars award.

ETS1-HMGA2 Axis Promotes Human Limbal Epithelial Stem Cell Proliferation.

This study aimed to investigate the role and molecular mechanism of ETS1 in the proliferation and differentiation of human limbal epithelial stem cells (LESCs). RNA-seq and quantitative real-time PCR were used to determine gene expression changes when ETS1 and HMGA2 was knocked down using short-hairpin RNAs or overexpressed by lentivirus. Immunofluorescence and flow cytometry experiments were performed to assess the roles of ETS1 and HMGA2 in LESC proliferation. ETS1-bound cis-regulatory elements and target genes in LESCs were identified using chromatin immunoprecipitation sequencing. The epigenetic features of ETS1-binding sites were assessed by the published histone modification and chromatin accessibility profiles. ETS1 was robustly expressed in LESCs but dramatically reduced on differentiation into corneal epithelial cells (CECs). ETS1 knockdown in LESCs inhibited cellular proliferation and activated CEC markers (KRT3, KRT12, CLU, and ALDH3A1). When ETS1 was overexpressed during CEC differentiation, LESC-associated genes were upregulated while CEC-associated genes were downregulated. The genome-wide binding profile of ETS1 was identified in LESCs. ETS1 occupied H3K4me3-marked promoters and H3K27ac/H3K4me1-marked enhancers. ETS1-binding sites were also enriched for chromatin accessibility signal. HMGA2 showed a consistent expression pattern with ETS1. ETS1 activates HMAG2 by binding to its promoter. Knockdown and overexpression experiments suggested that HMGA2 can promote LESC proliferation and inhibits its differentiation. ETS1 promotes LESC proliferation and inhibits its differentiation via activating HMGA2.

The lncRNA Tincr Regulates the Abnormal Differentiation of Intestinal Epithelial Stem Cells in the Diabetic State Via the miR-668-3p/Klf3 Axis.

Diabetes mellitus (DM) is among the most common chronic diseases, and diabetic enteropathy (DE), which is a complication caused by DM, is a serious health condition. Long noncoding RNAs (lncRNAs) are regulators of DE progression. However, the mechanisms of action of multiple lncRNAs involved in DE remain poorly understood. Reverse transcription-quantitative PCR (RT-qPCR) and in situ hybridization were used to analyze terminal differentiation-induced lncRNA (Tincr) expression in intestinal epithelial cells (IECs) in the DM state. Microarray analysis, bioinformatics analysis, and luciferase reporter assays were used to identify the genes targeted by Tincr. The role of miR-668-3p was then explored by up- and down-regulating its expression in vitro and in vivo. In this study, we observed that the level of lncRNA Tincr was increased in IECs in the DM state. More importantly, Tincr was associated with abnormal intestinal epithelial stem cell (IESC) differentiation in DM. Our mechanistic study demonstrated that Tincr is a major marker of Lgr5+ stem cells in DM. In addition, we investigated whether Tincr directly targets miR-668-3p and whether miR-668-3p targets Klf3. Our findings showed that Tincr sponged miR-668-3p, which attenuated abnormal IESC differentiation in DM by regulating Klf3 expression. This study presents evidence of an essential role for Tincr in IESC differentiation in DM.

Novel detection of stem cell niche within the stroma of limbus in the rabbit during postnatal development

Identifying and locating stem cell populations in the limbus may lead to developing a cell-based strategy for treating the corneal injury. Therefore, this study was the first to design a follow-up on the microscopical and histomorphometric changes in the rabbit limbus and to localize and demonstrate the limbal stem cell niche during postnatal development. The paraffin sections from the eyes of different postnatal-developmental stages were stained and examined using light microscopy. Furthermore, sections were immunohistologically stained for the epithelial stem cell differentiation marker, cytokeratin-14. Moreover, semithin and ultrathin sections were applied for ultrastructural demonstration of the stem cell niche. This study revealed that the number and thickness of limbal epithelial layers increased with age, whereas the thickness of limbal stroma decreased. Additionally, the immunohistochemical data showed that ck14 staining intensity increased in the limbal region where limbal stem cells reside. The semithin and ultrastructure investigation revealed stem cell clusters within the limbus’s underlying stroma close to the blood and nerve supply and surrounded by telocytes. Conclusively, isolated clusters of limbal epithelial stem cells combined with blood vessels, nerve fibers, and telocytes propose a harmonious microenvironment of a stem cell niche.

Gemini surfactant-based nanoparticles T-box1 gene delivery as a novel approach to promote epithelial stem cells differentiation and dental enamel formation.

Enamel is the highest mineralized tissue in the body protecting teeth from external stimuli, infections, and injuries. Enamel lacks the ability to self-repair due to the absence of enamel-producing cells in the erupted teeth. Here, we reported a novel approach to promote enamel-like tissue formation via the delivery of a key ameloblast inducer, T-box1 gene, into a rat dental epithelial stem cell line, HAT-7, using non-viral gene delivery systems based on cationic lipids. We comparatively assessed the lipoplexes prepared from glycyl-lysine-modified gemini surfactants and commercially available 1,2-dioleoyl-3-trimethylammonium-propane lipids at three nitrogen-to phosphate (N/P) ratios of 2.5, 5 and 10. Our findings revealed that physico-chemical characteristics and biological activities of the gemini surfactant-based lipoplexes with a N/P ratio of 5 provide the most optimal outcomes among those examined. HAT-7 cells were transfected with T-box1 gene using the optimal formulation then cultured in conventional 2D cell culture systems. Ameloblast differentiation, mineralization, bio-enamel interface and structure were assessed at different time points over 28days. Our results showed that our gemini transfection system provides superior gene expression compared to the benchmark agent, while keeping low cytotoxicity levels. T-box1-transfected HAT-7 cells strongly expressed markers of secretory and maturation stages of the ameloblasts, deposited minerals, and produced enamel-like crystals when compared to control cells. Taken together, our gemini surfactant-based T-box1 gene delivery system is effective to accelerate and guide ameloblastic differentiation of dental epithelial stem cells and promote enamel-like tissue formation. This study would represent a significant advance towards the tissue engineering and regeneration of dental enamel.

Co‐opting Lef‐1 and miR‐26bactivities to regulate dental epithelial stem cells and supporting tissues

ObjectiveStem cells are potent to self‐renew and differentiate which contribute to the regenerative process of various mature tissue types. It is important to understand how stem cell behaviors are regulated to give further insights to the application of stem cell therapies in tissue regeneration and repair. We use the developing murine lower incisor as a model to study stem cell maintenance and differentiation, which are housed in a niche and are fated to only a handful of differentiated cell types. Tooth development is a complex process involving rigid temporal and reciprocal regulation of transcription factors including Sox2 and Lef‐1, and signaling pathways including Wnt. We are particularly interested in the regulation between Lef‐1 and miR‐26b due to their inverse expression profiles during tooth development. Lef1 harbors a miR‐26b binding site in its 3’‐UTR and is negatively regulated by miR‐26b. In this study, we aim to understand the role of Lef‐1 and miR‐26b in dental epithelial stem cell (DESC) differentiation, determine the molecular mechanism of miR‐26b regulated Lef‐1 expression and identify genetic pathways for tooth development and regeneration.MethodsWith our newly developed miR‐26b over‐expression (miR‐26b OE) and Lef‐1 conditional over‐expression (COEL) mouse models, we determined the expression of miR‐26b and Lef‐1 by microarray and qPCR. To evaluate the phenotype of tooth development of these models, we performed H&E and IF staining during embryogenesis. Knowing the regulatory relationship between miR‐26b and Lef‐1, we crossed COEL and miR‐26b OE mice and evaluated their phenotype by microCT, H&E and IF staining to validate that COEL mouse phenotype could be rescued by miR‐26b OE. Because Lef‐1 is a Wnt signaling target, a Wnt signaling array was performed in miR‐26b OE E18.5 mandibles.ResultsThe COEL mouse exhibits tusk‐like incisors, a new stem cell compartment in the LaCL and increased DESC proliferation, whereas the miR‐26b OE mice do not have teeth. The expression of miR‐26b is decreased in the COEL mouse and Lef‐1 is decreased in miR‐26b OE mouse. The incisor phenotype observed in the COEL mouse can be rescued by crossing to the miR‐26b OE mouse. Other Wnt related genes including Wnt5a are downregulated whereas some Wnt related genes including Nanog are upregulated in miR‐26b OE mouse.ConclusionCOEL and miR‐26b OE mice have very different incisor phenotypes and crossing COEL and miR‐26b OE mouse rescues the phenotypes. These results further validate that miR‐26b regulates the dental epithelial stem cell niche by negatively regulating Lef‐1, which also indicate their important roles in tooth development. Wnt signaling participates in this regulatory network.SignificanceWith the first miR‐26b OE mouse model and COEL mouse model, we are the first to demonstrate a molecular mechanism for how stage specific miR‐26b expression acts to compartmentalize the stem cell niche through regulating the known developmental transcription factor Lef‐1. Our future direction is to determine the transcriptome and epigenome profiles of the DESC niche in WT, COEL and COEL x miR‐26b OE mice. We expect to identify new regulatory pathways using Lef‐1 and miR‐26b that can be used in bioengineering to repair or regenerate tissues.

The miR‐200family regulates the epithelial stem cell niche during craniofacial and dental development

Introduction & objective‐The murine lower incisor ectodermal organ contains a single epithelial stem cell niche that provides epithelial progenitor cells to the continuously growing rodent incisor. Calvaria sutures also contain stem cells that are regulated by specific factors. We have discovered that the microRNA (miR) ‐200 family is required for stem cell differentiation through targeting stem cell factors. We asked if the miR‐200 family regulated specific stem cell niches during development.Materials & methods‐ Our group used the plasmid‐based miR inhibitor system (PMIS) to knock down specific miR‐200 family members in mice and study their role during developmental processes. Our system inhibits one miR within a cluster to understand the role of that miR, which cannot be accomplished using gene targeting methods.Results‐We inhibited the miR‐200 family in developing murine embryos using new technology, resulting in an expanded epithelial stem cell niche and lack of cell differentiation. Inhibition of individual miRs within the miR‐200 cluster resulted in differential developmental and cell morphology defects. miR‐200 inhibition increased the expression of dental epithelial stem cell markers, expanded the stem cell niche and decreased progenitor cell differentiation. RNA‐seq. identified miR‐200 regulatory pathways involved in cell differentiation and compartmentalization of the stem cell niche. The miR‐200 family regulates signaling pathways required for cell differentiation and cell cycle progression. New miR‐200targets demonstrate gene networks and pathways controlling cell differentiation and maintenance of the stem cell niche.Conclusion‐This is the first report demonstrating how the miR‐200 family is required for in vivo progenitor cell proliferation and differentiation.Significance/implication‐This new approach has identified several new scientific advancements in stem cell niche biology. 1) miR‐200 family members are required for in vivo epithelial stem cell differentiation and stem cell niche maintenance; 2) The miR‐200 family regulates genes required for generating the different dental epithelial cell lineages; 3) miR‐200expression may be a common mechanism for defining the boundaries of adult epithelial stem cell niches; 4) By inhibiting the miRs that become active when a progenitor cell becomes fated to differentiate, the in vivo cell differentiation programs are determined assuming their terminal fates; 5) Dental epithelial stem cells and suture stem cells are regulated by members of the miR‐200 family to yield the differentiated progeny and determine cell fate.

BMP feed-forward loop promotes terminal differentiation in gastric glands and is interrupted by H. pylori-driven inflammation

Helicobacter pylori causes gastric inflammation, gland hyperplasia and is linked to gastric cancer. Here, we studied the interplay between gastric epithelial stem cells and their stromal niche under homeostasis and upon H. pylori infection. We find that gastric epithelial stem cell differentiation is orchestrated by subsets of stromal cells that either produce BMP inhibitors in the gland base, or BMP ligands at the surface. Exposure to BMP ligands promotes a feed-forward loop by inducing Bmp2 expression in the epithelial cells themselves, enforcing rapid lineage commitment to terminally differentiated mucous pit cells. H. pylori leads to a loss of stromal and epithelial Bmp2 expression and increases expression of BMP inhibitors, promoting self-renewal of stem cells and accumulation of gland base cells, which we mechanistically link to IFN-γ signaling. Mice that lack IFN-γ signaling show no alterations of BMP gradient upon infection, while exposure to IFN-γ resembles H. pylori-driven mucosal responses.

Bmi1 signaling maintains the plasticity of airway epithelial progenitors in response to persistent silica exposures

Silicosis is caused by the continuous inhalation of environmental silica dust. The repetitive exposure of silica induces airway epithelial cell injury, leads proliferative exhaustion of epithelial stem cells, ultimately results in the lung remodeling and the development of silicosis. The B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi1) is a pivotal transcription factor in stem cell self-renewal and proliferation of many tissues including the lung, but its role in the airway basal cell proliferation and differentiation during the pathogenesis of silicosis in lung has yet been investigated. In this study, the function of Bmi1 in airway basal cell proliferation and differentiation in response to silica challenge was investigated in lungs of silicosis mice and primary human bronchial epithelia cells (HBECs) exposed to silica dioxide (SiO2). Results showed a decreased expression of Bmi1 protein, epithelial basal cell markers Krt14 and Krt5, club cell marker Clara cell secretory protein, and ciliated cell marker acetyl-α-tubulin in silicosis lungs, compared to healthy mice. In consistence, a persistent exposure of SiO2 reduced the capacity of cell proliferation and differentiation in HBECs, as ascertained by the reduction of differentiated epithelial cell markers and BMI1 expression, while an increased P21-positive senescent cell fraction. Moreover, an overexpression of BMI1 in HBECs reduced the SiO2-senescent cells, enhanced the potency of cell proliferation and differentiation, and increased capacity of airway epithelial regeneration in response to the persistent exposure of SiO2. These data suggest that Bmi1 is a key transcription factor engaging in maintaining the self-renewal, proliferation and differentiation of epithelial stem cells in lung during the development of silicosis disease.

Generation and Characterization of Murine Oral Mucosal Organoid Cultures.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

HC-HA/PTX3 from amniotic membrane reverts senescent limbal niche cells to Pax6+ neural crest progenitors to support limbal epithelial progenitors

Quiescence and self‐renewal of human corneal epithelial progenitor/stem cells (LEPC) are regulated by the limbal niche, presumably through close interaction with limbal (stromal) niche cells (LNC). Paired box homeotic gene 6 (Pax6), a conserved transcription factor essential for eye development, is essential for proper differentiation of limbal and corneal epithelial stem cells. Pax6 haploinsufficiency causes limbal stem cell deficiency, which leads to subsequent corneal blindness. We previously reported that serial passage of nuclear Pax6+ LNC resulted in the gradual loss of nuclear Pax6+ and neural crest progenitor status, the latter of which was reverted upon recovery of Pax6. These findings suggest Pax6 plays a pivotal role in supporting the self‐renewal of LEPC in limbal niche. Herein, we show that HC‐HA/PTX3, a unique matrix purified from amniotic membrane (AM) and consists of heavy chain 1of inter‐α‐trypsin inhibitor covalently linked to hyaluronic acid and complexed with pentraxin 3, is capable of reverting senescent LNC to nuclear Pax6+ neural crest progenitors that support self‐renewal of LEPC. Such reversion is causally linked to early cell aggregation mediated by activation of C‐X‐C chemokine receptor type 4 (CXCR4)‐mediated signaling followed by activation of bone morphogenetic protein (BMP) signaling. Furthermore, CXCR4‐mediated signaling, but not BMP signaling, controls recovery of the nuclear Pax6+ neural crest progenitors. These findings not only explain why AM helps in vivo and ex vivo expansion of human LEPC, but they also illuminate the potential role of HC‐HA/PTX3 as a surrogate matrix niche that complements stem cell‐based therapies in regenerative medicine.

β-Catenin/Lin28/let-7 regulatory network determines type II alveolar epithelial stem cell differentiation phenotypes following thoracic irradiation.

The contribution of type II alveolar epithelial stem cells (AEC II) to radiation-induced lung fibrosis (RILF) is largely unknown. Cell differentiation phenotypes are determined by the balance between Lin28 and lethal-7 microRNA (let-7 miRNA). Lin28 is activated by β-catenin. The aim of this study was to track AEC II phenotypes at different phases of injury following thoracic irradiation and examine the expression of β-catenin, Lin28 and let-7 to identify their role in AEC II differentiation. Results showed that coexpression of prosurfactant protein C (proSP-C, an AEC II biomarker) and HOPX (homeobox only protein X, an AEC I biomarker) or vimentin (a differentiation marker) was detected in AEC II post-irradiation. The protein expression levels of HOPX and proSP-C were significantly downregulated, but vimentin was significantly upregulated following irradiation. The expression of E-cadherin, which prevents β-catenin from translocating to the nucleus, was downregulated, and the expression of β-catenin and Lin28 was upregulated after irradiation (P < 0.05 to P < 0.001). Four let-7 miRNA members (a, b, c and d) were upregulated in irradiated lungs (P < 0.05 to P < 0.001), but let-7d was significantly downregulated at 5 and 6 months (P < 0.001). The ratios of Lin28 to four let-7 members were low during the early phase of injury and were slightly higher after 2 months. Intriguingly, the Lin28/let-7d ratio was strikingly increased after 4 months. We concluded that β-catenin contributed to RILF by promoting Lin28 expression, which increased the number of AEC II and the transcription of profibrotic molecules. In this study, the downregulation of let-7d miRNA by Lin28 resulted in the inability of AEC II to differentiate into type I alveolar epithelial cells (AEC I).

Electrospun PLGA Fiber Diameter and Alignment of Tendon Biomimetic Fleece Potentiate Tenogenic Differentiation and Immunomodulatory Function of Amniotic Epithelial Stem Cells.

Injured tendons are challenging in their regeneration; thus, tissue engineering represents a promising solution. This research tests the hypothesis that the response of amniotic epithelial stem cells (AECs) can be modulated by fiber diameter size of tendon biomimetic fleeces. Particularly, the effect of electrospun poly(lactide-co-glycolide) (PLGA) fleeces with highly aligned microfibers possessing two different diameter sizes (1.27 and 2.5 µm: ha1- and ha2-PLGA, respectively) was tested on the ability of AECs to differentiate towards the tenogenic lineage by analyzing tendon related markers (Collagen type I: COL1 protein and mRNA Scleraxis: SCX, Tenomodulin: TNMD and COL1 gene expressions) and to modulate their immunomodulatory properties by investigating the pro- (IL-6 and IL-12) and anti- (IL-4 and IL-10) inflammatory cytokines. It was observed that fiber alignment and not fiber size influenced cell morphology determining the morphological change of AECs from cuboidal to fusiform tenocyte-like shape. Instead, fleece mechanical properties, cell proliferation, tenogenic differentiation, and immunomodulation were regulated by changing the ha-PLGA microfiber diameter size. Specifically, higher DNA quantity and better penetration within the fleece were found on ha2-PLGA, while ha1-PLGA fleeces with small fiber diameter size had better mechanical features and were more effective on AECs trans-differentiation towards the tenogenic lineage by significantly translating more efficiently SCX into the downstream effector TNMD. Moreover, the fiber diameter of 1.27 µm induced higher expression of pro-regenerative, anti-inflammatory interleukins mRNA expression (IL-4 and IL-10) with favorable IL-12/IL-10 ratio with respect to the fiber diameter of 2.5 µm. The obtained results demonstrate that fiber diameter is a key factor to be considered when designing tendon biomimetic fleece for tissue repair and provide new insights into the importance of controlling matrix parameters in enhancing cell differentiation and immunomodulation either for the cells functionalized within or for the transplanted host tissue.

Tight Junction Protein Claudin-7 Is Essential for Intestinal Epithelial Stem Cell Self-Renewal and Differentiation

Tight Junction Protein Claudin-7 Is Essential for Intestinal Epithelial Stem Cell Self-Renewal and Differentiation

Tight Junction Protein Claudin-7 Is Essential for Intestinal Epithelial Stem Cell Self-Renewal and Differentiation

Background & AimsClaudin-7 (Cldn7) is a tight junction (TJ) membrane protein located at the apical TJ and basolateral side of intestinal epithelial cells. Deletion of Cldn7 by gene targeting leads to the inflammatory bowel disease–like phenotype in mice, which includes weight loss, diarrhea, mucosa ulceration, and severe intestinal epithelial damage. In this study, we test our hypothesis that Cldn7 plays a critical role in regulating intestinal crypt stem cell functions.MethodsGene expression microarray, quantitative reverse-transcription polymerase chain reaction, in situ hybridization, histologic examinations, immunoblotting, 3-dimensional organoid culture, and various treatments to rescue Cldn7-deficient organoid defects were conducted using global Cldn7 knockout mice and inducible, conditional Cldn7 knockout mice.ResultsGene deletion of Cldn7 in intestines showed significant alteration of expression profiles with striking down-regulation of intestinal crypt stem cell markers such as Olfm4, dislocated proliferative cells, and disrupted epithelial cell differentiation. In addition, the isolated Cldn7-deficient crypts where the stem cells reside were either unable to survive at all or formed defective spheroids, highlighting the functional impairment of crypt stem cells in the absence of Cldn7. Remarkably, the Cldn7-expressing organoids with buddings underwent rapid cell degeneration within days after turning off Cldn7 expression in the culture. We identified that activation of Wnt/β-catenin signaling rescued the organoid defects caused by Cldn7 deletion.ConclusionsIn this study, we show that Cldn7 is indispensable in controlling Wnt/β-catenin signaling–dependent intestinal epithelial stem cell survival, self-renewal, and cell differentiation. This study could open a door to study roles of TJ proteins in stem cell regulations in other tissues and organs.

Evolutionary history of the MTG gene family in vertebrates

The highly conservedMTGgene family includes three homologs in vertebrates (MTG8,MTGR1,MTG16) encoding transcriptional corepressors, which are important in haemopoiesis, neurogenesis and epithelial stem cell differentiation. These genes are of particular interest because they are involved in translocations, associated with different types of cancer. Looking at how this gene family evolved might provide insights into history of its structural and functional diversification. We have performed a phylogenetic analysis ofMTGnucleotide and protein sequences to examine the evolutionary events. The domain organization ofMTGgene products was clarified, the mechanism of appearance of the firstMTGgene was revealed and the ancestor taxon was determined. Also the mechanism ofMTGgene family emergence was established. In addition, analysis of the rates of evolution acting on individual domains was made, and conservative positions within each gene ofMTGfamily were determined.