Markers Of Epithelial Cell Differentiation Research Articles

Lipid signature associated with chronic colon inflammation reveals a dysregulation in colonocyte differentiation process

Inflammatory Bowel Disease (IBD) comprises a heterogeneous group of chronic inflammatory conditions of the gastrointestinal tract that include ulcerative colitis (UC) and Crohn's disease. Although the etiology is not well understood, IBD is characterized by a loss of the normal epithelium homeostasis that disrupts the intestinal barrier of these patients. Previous work by our group demonstrated that epithelial homeostasis along the colonic crypts involves a tight regulation of lipid profiles. To evaluate whether lipidomic profiles conveyed the functional alterations observed in the colonic epithelium of IBD, we performed matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) analyses of endoscopic biopsies from inflamed and non-inflamed segments obtained from UC patients. Our results indicated that lipid profiling of epithelial cells discriminated between healthy and UC patients. We also demonstrated that epithelial cells of the inflamed mucosa were characterized by a decrease in mono- and di-unsaturated fatty acid-containing phospholipids and higher levels of arachidonic acid-containing species, suggesting an alteration of the lipid gradients occurring concomitantly to the epithelial differentiation. This result was reinforced by the immunofluorescence analysis of EPHB2 and HPGD, markers of epithelial cell differentiation, sustaining that altered lipid profiles were at least partially due to a faulty differentiation process. Overall, our results showed that lipid profiling by MALDI-MSI faithfully conveys molecular and functional alterations associated with the inflamed epithelium, providing the foundation for a novel molecular characterization of UC patients.

Vitamin D restriction and/or a high-fat diet influence intestinal alkaline phosphatase activity and serum endotoxin concentration, increasing the risk of metabolic endotoxemia in rats

Vitamin D insufficiency induces calcification disorder of bone or a decrease in bone mineral density, increasing the risk of fracture. Alkaline phosphatase (ALP) activity, a differentiation marker for intestinal epithelial cells, is regulated by vitamin D. It has also been suggested that ALP may prevent metabolic endotoxemia by dephosphorylating lipopolysaccharide. We hypothesized that vitamin D restriction and/or a high-fat diet influences ALP activity in each tissue and serum lipopolysaccharide concentrations and increases the risk of metabolic endotoxemia. Eleven-week-old female rats were divided into 4 groups: basic control diet (Cont.), basic control diet with vitamin D restriction (DR), high-fat diet (HF), and high-fat diet with vitamin D restriction (DRHF) groups. They were acclimated for 28 days. The results of 2-way analysis of variance showed that intestinal ALP activity, which may contribute to an improvement in phosphate/lipid metabolism and longevity, in the high-fat diet groups (HF and DRHF) was higher than in the low-fat diet groups (Cont. and DR). ALP activity in the vitamin D-restricted groups (DR and DRHF) was lower than in the vitamin D-sufficient groups (Cont. and HF). Furthermore, serum endotoxin concentrations were significantly higher in the high-fat diet groups (HF and DRHF) than in the low-fat diet groups (Cont. and DR). In the vitamin D-restricted groups (DR and DRHF), serum endotoxin concentrations were also significantly higher than in the vitamin D-sufficient groups (Cont. and HF). These results suggest that vitamin D restriction and/or a high-fat diet increases the risk of metabolic endotoxemia.

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.

Organ-specific extracellular matrix directs trans-differentiation of mesenchymal stem cells and formation of salivary gland-like organoids in vivo

BackgroundCurrent treatments for salivary gland (SG) hypofunction are palliative and do not address the underlying cause or progression of the disease. SG-derived stem cells have the potential to treat SG hypofunction, but their isolation is challenging, especially when the tissue has been damaged by disease or irradiation for head and neck cancer. In the current study, we test the hypothesis that multipotent bone marrow-derived mesenchymal stem cells (BM-MSCs) in a rat model are capable of trans-differentiating to the SG epithelial cell lineage when induced by a native SG-specific extracellular matrix (SG-ECM) and thus may be a viable substitute for repairing damaged SGs.MethodsRat BM-MSCs were treated with homogenates of decellularized rat SG-ECM for one hour in cell suspension and then cultured in tissue culture plates for 7 days in growth media. By day 7, the cultures contained cell aggregates and a cell monolayer. The cell aggregates were hand-selected under a dissecting microscope, transferred to a new tissue culture dish, and cultured for an additional 7 days in epithelial cell differentiation media. Cell aggregates and cells isolated from the monolayer were evaluated for expression of SG progenitor and epithelial cell specific markers, cell morphology and ultrastructure, and ability to form SG-like organoids in vivo.ResultsThe results showed that this approach was very effective and guided the trans-differentiation of a subpopulation of CD133-positive BM-MSCs to the SG epithelial cell lineage. These cells expressed amylase, tight junction proteins (Cldn 3 and 10), and markers for SG acinar (Aqp5 and Mist 1) and ductal (Krt 14) cells at both the transcript and protein levels, produced intracellular secretory granules which were morphologically identical to those found in submandibular gland, and formed SG-like organoids when implanted in the renal capsule in vivo.ConclusionsThe results of this study suggest the feasibility of using autologous BM-MSCs as an abundant source of stem cells for treating SG hypofunction and restoring the production of saliva in these patients.

Establishment and characterization of a sheep endometrial epithelial cell line

Endometrial epithelial cells play a significant role in the "dialogue" between the embryo and the mother, but in vitro studies to clarify this are hampered by the limited lifespan of primary cells. As such, it is necessary to develop an in vitro model to study endometrial function. Morphological analysis showed that the pEECs were homogeneous, formed characteristic cobblestone monolayers, and expressed the epithelial cell-specific marker, cytokeratin 18. The isolated and purified cells were transfected with a plasmid encoding human telomerase reverse transcriptase (TERT) gene, pCI-neo-TERT, to establish an immortal endometrial epithelial cell line (iEECs). The transfected cells were cultured with G418 and monoclonal cells were selected for expanded culture. Expression of TERT mRNA was detected by RT-qPCR and protein was quantitated by Western blot. TERT expression was stable and continued to be active with no signs of aging. Assays for cell proliferation and apoptosis indicated higher proliferation and cellular activity in iEECs than pEECs. After stimulated by interferon tau (IFN-τ), both iEECs and pEECs showed similar upregulation levels in all the underlying genes. Taken together, these findings demonstrate that iEECs retained the basic morphology and function of pEECs, providing a robust in vitro model for study of the function of ovine endometrial epithelial cells.

Pseudomonas aeruginosa Affects Airway Epithelial Response and Barrier Function During Rhinovirus Infection

Cystic fibrosis (CF) lung disease is aggravated by recurrent and ultimately chronic bacterial infections. One of the key pathogens in adult CF lung disease is P. aeruginosa (PA). In addition to bacteria, respiratory viral infections are suggested to trigger pulmonary exacerbations in CF. To date, little is known on how chronic infections with PA influence susceptibility and response to viral infection. We investigated the interactions between PA, human rhinovirus (HRV) and the airway epithelium in a model of chronic PA infection using differentiated primary bronchial epithelial cells (pBECs) and clinical PA isolates obtained from the respiratory sample of a CF patient. Cells were repeatedly infected with either a mucoid or a non-mucoid PA isolate for 16 days to simulate chronic infection, and subsequently co-infected with HRV. Key cytokines and viral RNA were quantified by cytometric bead array, ELISA and qPCR. Proteolytic degradation of IL-6 was analyzed by Western Blots. Barrier function was assessed by permeability tests and transepithelial electric resistance measurements. Virus infection stimulated the production of inflammatory and antiviral mediators, including interleukin (IL)-6, CXCL-8, tumor necrosis factor (TNF)-α, and type I/III interferons. Co-infection with a non-mucoid PA isolate increased IL-1β protein concentrations (28.88 pg/ml vs. 6.10 pg/ml), but in contrast drastically diminished levels of IL-6 protein (53.17 pg/ml vs. 2301.33 pg/ml) compared to virus infection alone. Conditioned medium obtained from co-infections with a non-mucoid PA isolate and HRV was able to rapidly degrade recombinant IL-6 in a serine protease-dependent manner, whereas medium from individual infections or co-infections with a mucoid isolate had no such effect. After co-infection with HRV and the non-mucoid PA isolate, we detected lower mRNA levels of Forkhead box J1 (FOXJ1) and Cilia Apical Structure Protein (SNTN), markers of epithelial cell differentiation to ciliated cells. Moreover, epithelial permeability was increased and barrier function compromised compared to single infections. These data show that PA infection can influence the response of bronchial epithelial cells to viral infection. Altered innate immune responses and compromised epithelial barrier function may contribute to an aggravated course of viral infection in PA-infected airways.

High-Grade Sarcoma Arising in the Background of Growing Teratoma Syndrome; A Rare Case

Abstract Introduction/Objective Growing teratoma syndrome is a rare condition seen in patients with a history of immature teratomas or mixed germ cell tumor status post-treatment. It usually occurs within the first two years of diagnosis but rarely can be seen years later. Methods/Case Report We report a case of a 48-year-old female with a previous history of ovarian malignancy in adolescence status post-TAH/BSO and chemotherapy, now presenting with a 23 cm multi cystic septate mass with some solid components in the subdiaphragmatic area, extending into the liver on imaging. Nodular implants along the surface of the right hepatic lobe and multiple peritoneal implants were seen. Histology of peritoneal implants showed fibro adipose tissue admixed with rare scattered glandular elements, epithelial fragments, and mature neuroglial tissue, consistent with mature teratoma. Liver biopsy revealed short fascicles of spindled, epithelioid and rhabdoid tumor cells admixed with foci of the myxoid stroma. Scattered moderate cytologic atypia, atypical mitosis, and necrosis were appreciated. Tumor cells showed strong and diffuse reactivity to vimentin while being negative for epithelial, neural/mesenchymal, mesothelial, sex cord-stromal, and germ cell differentiation markers. Differential diagnostic considerations included spindle cell transformation of a germ cell tumor or high-grade sarcoma, not otherwise specified arising in the background of growing teratoma syndrome. FISH testing for isochromosome 12p was negative excluding sarcomatoid germ cell tumor. Results (if a Case Study enter NA) NA Conclusion This case study highlights the importance of considering the rare complication of high-grade sarcoma arising in the background of growing teratoma syndrome.

Recombinant soluble thrombomodulin accelerates epithelial stem cell proliferation in mouse intestinal organoids and promotes the mucosal healing in colitis.

Epithelial regeneration, a critical step for the mucosal healing in inflammatory bowel disease, is tightly regulated by stem cells. Therefore, identification of the specific factors that induce stem cell proliferation could contribute to the development of effective strategies for treating inflammatory bowel disease. Recombinant soluble thrombomodulin (rsTM) has previously been shown to promote cell proliferation in skin and corneal wound healing in murine models, but its effects on intestinal epithelial cell proliferation remains unclear. Mouse intestinal organoids and dextran sulfate sodium (DSS)-induced colitis mouse model were used to assess the effects of rsTM on proliferation of intestinal epithelial cells. The size and budding morphologies of organoids were studied by confocal microscopy. The gene expression levels were analyzed by quantitative real-time polymerase chain reaction and immunofluorescence analysis. The effects of rsTM on DSS-induced colitis were investigated by evaluating body weight changes, colon length, histological score, and survival rate. The rsTM markedly stimulated the growth of intestinal organoids, thereby increasing the surface areas and budding phenotypes of the organoids. rsTM also significantly upregulated the gene expression of intestinal stem cell-specific and epithelial cell-specific markers in a dose-dependent manner. Furthermore, the treatment with high concentrations of rsTM significantly improved the recovery of body weight, histological outcomes, colon length shortening, and prolonged the survival of mice with colitis. The rsTM promotes intestinal stem cell proliferation in intestinal organoids and enhances the mucosal healing during recovery phase in DSS-induced colitis.

The effect of 4-hexylresocinol administration on SCC-9 cells: mass spectrometric identification of proteins and cDNA microarray analysis

BackgroundIn stress situations, bacteria produce dormancy-inducing factors to stop cell growth. The dormancy-inducing factors may have an inhibitory effect on tumor cell growth. Here we analyzed the differentially expressed protein profiles after 4-hexylresorcinol (4HR), one of the dormancy-inducing factors, administration using in vitro oral squamous carcinoma cells (SCC-9).MethodThe control group was SCC-9 cells culture without 4HR administration. The experimental group received 10 μg/mL of 4HR. Collected proteins from each group were loaded for 2D electrophoresis. Among the separated proteins, 20 differentially expressed proteins were selected and processed for LC-MS/MS.ResultsIn proteomic analysis, the expression of keratin 1, keratin 10, and histone H2B were increased. In cDNA microarray assay, the genes related to the cellular differentiation (involucrin, keratin 13, 14) were highly expressed in the 4HR treated group (fold ratio > 2.0; Table 2). Interestingly, histone family was upregulated in the cDNA microarray assay.ConclusionThe administration of 4HR on SCC-9 cells increased epithelial cell differentiation markers and histone.

Epigenetic Effects of Blackberry Extract on Human Colorectal Cancer Cells

Fruit-derived polyphenolic compounds have been shown to exert anticancer effects via epigenetic mechanisms. In this study, we investigated the effect of blackberry extract on the expression of DNMTs (Dnmt1, Dnmt3a, and Dnmt3b) and HDACs (HDAC1-4 and SIRT1) and its influence on the cellular differentiation and promoter DNA methylation of tumor-related genes using a panel of six human CRC cell lines. Treatment with IC20 and IC50 concentrations of blackberry extract for 72 h significantly reduced Dnmt1 and Dnmt3b transcript levels in HCT116, SW480, HT29/219, SW742, and LS180 cells in a dose-dependent manner. Blackberry also induced promoter DNA demethylation of SFRP2 and p16 genes in four tested CRC cell lines. Berry treatment, however, upregulated Dnmt3a genes in SW480, SW742, and HT29/219 cell lines. A dose-dependent and cell-type-specific reduction of HDAC1, HDAC2, and HDAC4 expressions were observed in CRC-treated cells. Treatment with berry extract induced the expression of SIRT1 gene in HCT116 and HT29/219 cells and increased the expression of two colonic epithelial cell differentiation markers, carcinoembryonic antigen (CEA) and alkaline phosphatase in LS180 cells in a time-dependent manner. This study is the first to report the epigenetic effects of blackberry in cancer cells.

Pretreatment of aged mice with retinoic acid supports alveolar regeneration via upregulation of reciprocal PDGFA signalling

ObjectivesIdiopathic pulmonary fibrosis (IPF) primarily affects the aged population and is characterised by failure of alveolar regeneration, leading to loss of alveolar type 1 (AT1) cells. Aged mouse models of...

Biomimetic sulfated glycosaminoglycans maintain differentiation markers of breast epithelial cells and preferentially inhibit proliferation of cancer cells

Glycosaminoglycans (GAG) are key elements involved in various physiological and pathological processes including cancer. Several GAG-based drugs have been developed showing significant results and potential use as cancer therapeutics. We previously reported that alginate sulfate (AlgSulf), a GAG-mimetic, reduces the proliferation of lung adenocarcinoma cells. In this study, we evaluated the preferential effect of AlgSulf on tumorigenic and nontumorigenic mammary epithelial cells in 2D, 3D, and coculture conditions. AlgSulf were synthesized with different degrees of sulfation (DSs) varying from 0 to 2.7 and used at 100 µg/mL on HMT-3522 S1 (S1) nontumorigenic mammary epithelial cells and their tumorigenic counterparts HMT-3522 T4–2 (T4–2) cells. The anti-tumor properties of AlgSulf were assessed using trypan blue and bromodeoxyuridine proliferation (BrdU) assays, immunofluorescence staining and transwell invasion assay. Binding of insulin and epidermal growth factor (EGF) to sulfated substrates was measured using QCM-D and ELISA. In 2D, the cell growth rate of cells treated with AlgSulf was consistently lower compared to untreated controls (p<0.001) and surpassed the effect of the native GAG heparin (positive control). In 3D, AlgSulf preferentially hindered the growth rate and the invasion potential of tumorigenic T4–2 nodules while maintaining the formation of differentiated polarized nontumorigenic S1 acini. The preferential growth inhibition of tumorigenic cells by AlgSulf was confirmed in a coculture system (p<0.001). In the ELISA assay, a trend of EGF binding was detected for sulfated polysaccharides while QCM-D analysis showed negligible binding of insulin and EGF to sulfated substrates. The preferential effect mediated by the mimetic sulfated GAGs on cancer cells may in part be growth factor dependent. Our findings suggest a potential anticancer therapeutic role of AlgSulf for the development of anticancer drugs.

Biomimetic Sulfated GAGs Maintain Differentiation Markers of Breast Epithelial Cells and Preferentially Inhibit Proliferation of Cancer Cells

Glycosaminoglycans (GAG) are key elements involved in various physiological and pathological processes including cancer. Several GAG-based drugs have been developed showing significant results and potential use as cancer therapeutics. We previously reported that alginate sulfate (AlgSulf), a GAG-mimetic, reduces the proliferation of lung adenocarcinoma cells. In this study, we evaluated the preferential effect of AlgSulf on tumorigenic and nontumorigenic mammary epithelial cells in 2D, 3D, and coculture conditions. AlgSulf were synthesized with different degrees of sulfation (DSs) varying from 0 to 2.7 and used at 100 µg/mL on HMT-3522 S1 (S1) nontumorigenic mammary epithelial cells and their tumorigenic counterparts HMT-3522 T4-2 (T4-2) cells. The anti-tumor properties of AlgSulf were assessed using trypan blue assay, immunofluorescence staining and transwell invasion assay. Binding of insulin and epidermal growth factor (EGF) to sulfated substrates was measured using QCM-D and ELISA. In 2D, the cell growth rate of cells treated with AlgSulf was consistently lower compared to untreated controls (p<0.001) and surpassed the effect of the native GAG heparin (positive control). In 3D, AlgSulf preferentially hindered the growth rate and the invasion potential of tumorigenic T4-2 nodules while maintaining the formation of differentiated polarized nontumorigenic S1 acini. The preferential growth inhibition of tumorigenic cells by AlgSulf was confirmed in a coculture system (p<0.001). In QCM-D and ELISA binding studies, negligible binding of insulin and EGF to sulfated substrates was reported. The preferential effect mediated by the mimetic sulfated GAGs on cancer cells seems to be independent of growth factors binding. Our findings suggest a potential anticancer therapeutic role of AlgSulf for the development of anticancer drugs.

S1P Lyase siRNA Dampens Malignancy of DLD-1 Colorectal Cancer Cells.

Sphingosine-1-phosphate lyase 1 (S1P lyase or SGPL1) is an essential sphingosine-1-phosphate-degrading enzyme. Its manipulation favors onset and progression of colorectal cancer and others in vivo. Thus, SGPL1 is an important modulator of cancer initiation. However, in established cancer, the impact of retrospective SGPL1 modulation is elusive. Herein, we analyzed how SGPL1 siRNA affects malignancy of the human colorectal cancer cells DLD-1 and found that in parallel to the reduction of SGPL1 expression levels, migration, invasion, and differentiation status changed. Diminished SGPL1 expression was accompanied with reduced cell migration and cell invasion in scratch assays and transwell assays, whereas metabolic activity and proliferation was not altered. Decreased migration was attended by increased cell-cell-adhesion through upregulation of E-cadherin and formation of cadherin-actin complexes. Spreading cell islets showed lower vimentin abundance in border cells. Furthermore, SGPL1 siRNA treatment induced expression of epithelial cell differentiation markers, such as intestinal alkaline phosphatase and cytokeratin 20. Hence, interference with SGPL1 expression augmented a partial redifferentiation of colorectal cancer cells toward normal colon epithelial cells. Our investigation showed that SGPL1 siRNA influenced tumorigenic activity of established colorectal cancer cells. We therefore suggest SGPL1 as a target for lowering malignant potential of already existing cancer.

A composite Gelatin/hyaluronic acid hydrogel as an ECM mimic for developing mesenchymal stem cell-derived epithelial tissue patches.

Here we report fabrication of Gelatin-based biocomposite films and their application in developing epithelial patches. The films were loaded with an epithelial cell growth factor cocktail and used as an extracellular matrix mimic for in vitro regeneration of organized respiratory epithelium using Calu-3 cell line and mesenchymal stem cells (MSCs). Our data show differentiation of Calu-3 cells on composite films as evidenced by tight junction protein expression and barrier formation. The films also supported attachment, migration, and proliferation of alveolar basal epithelial cell line A549. We also show the suitability of the composite films as a biomimetic scaffold and growth factor delivery platform for differentiation of human MSCs to epithelial cells. MSCs differentiation to the epithelial lineage was confirmed by staining for epithelial and stem cell specific markers. Our data show that the MSCs acquire the epithelial characteristics after 2 weeks with significant reduction in vimentin, increase in pan cytokeratin expression, and morphological changes. However, despite the expression of epithelial lineage markers, these cells did not form fully functional tight junctions as evidenced by low expression of junctional protein ZO1. Further optimisation of culture conditions and growth factor cocktail is required to enhance tight junction formation in MSCs-derived epithelial cells on the composite hydrogels. Nevertheless, our data clearly highlight the possibility of using MSCs in epithelial tissue engineering and the applicability of the composite hydrogels as transferrable extracellular matrix mimics and delivery platforms with potential applications in regenerative medicine and in vitro modelling of barrier tissues.

Notch signalling is a potential resistance mechanism of progenitor cells within patient-derived prostate cultures following ROS-inducing treatments.

Low Temperature Plasma (LTP) generates reactive oxygen and nitrogen species, causing cell death, similarly to radiation. Radiation resistance results in tumour recurrence, however mechanisms of LTP resistance are unknown. LTP was applied to patient‐derived prostate epithelial cells and gene expression assessed. A typical global oxidative response (AP‐1 and Nrf2 signalling) was induced, whereas Notch signalling was activated exclusively in progenitor cells. Notch inhibition induced expression of prostatic acid phosphatase (PAP), a marker of prostate epithelial cell differentiation, whilst reducing colony forming ability and preventing tumour formation. Therefore, if LTP is to be progressed as a novel treatment for prostate cancer, combination treatments should be considered in the context of cellular heterogeneity and existence of cell type‐specific resistance mechanisms.

Puberty is a critical window for the impact of diet on mammary gland development in the rabbit.

BackgroundNutritional changes can affect future lactation efficiency. In a rabbit model, an obesogenic diet initiated before puberty and pursued throughout pregnancy enhances mammary differentiation, but when started during the neonatal period can cause abnormal mammary development in early pregnancy. The aim of this study was to investigate the impact of an unbalanced diet administered during the pubertal period only.ResultsConsuming an obesogenic diet at puberty did not affect either metabolic parameters or certain maternal reproductive parameters at the onset of adulthood. In contrast, at Day 8 of pregnancy, epithelial tissue showed a lower proliferation rate in obesogenic‐diet fed rabbits than in control‐diet fed rabbits. Wap and Cx26 genes, mammary epithelial cell differentiation markers, were upregulated although Wap protein level remained unchanged. However, the expression of genes involved in lipid metabolism and in alveolar formation was not modified.ConclusionTaken together, our results demonstrate that the consumption for 5 weeks of an obesogenic diet during the pubertal period initiates mammary structure modifications and affects mammary epithelial cell proliferation and differentiation. Our findings highlight the potentially important role played by unbalanced nutrition during critical early‐life windows in terms of regulating mammary epithelial cell differentiation and subsequent function in adulthood.

Induction of Mouse Peritoneum Mesenchymal Stem Cells into Germ Cell-Like Cells Using Follicular Fluid and Cumulus Cells-Conditioned Media.

The peritoneum mesothelium lines body cavities and has the same origin as ovarian surface epithelium with probable existence of peritoneum mesenchymal stem cells (PMSCs). In the present research, PMSCs were isolated from peritoneum and differentiated into ovarian cell-like cells using human follicular fluid (HFF) and human cumulus-conditioned medium (HCCM). Anterior abdominal wall and intestinal peritoneum explants were used for cells isolation and cultured in Dulbecco's modified Eagle's medium. After passage 3, purified PMSCs were assessed for morphology, proliferation rate, and cell viability. Then, isolated PMSCs underwent two characterization procedures: (1) differentiation to mesodermal lineage and (2) expression of mesenchymal (CD90 and CD44) and epithelial cell (CK19) markers. The characterized PMSCs were differentiated into ovarian cell-like cells using 10% HFF and 50% HCCM for 21 days, and the expressions of oocyte (Zp3, Gdf9), germ cell (Ddx4, Dazl), granulosa cell (Amh), and theca cell (Lhr) markers were assessed using real-time polymerase chain reaction and immunocytofluorescence assay. Both anterior abdominal wall and intestinal peritoneum mesenchymal stem cells (AP-MSCs and IP-MSCs) showed mesenchymal characters and differentiated to adipocyte and osteocyte. AP-MSCs expressed mesenchymal- and epithelial cell-specific markers more than IP-MSCs and showed an analytically better proliferation rate. The induced AP-MSCs and IP-MSCs were expressed as germ and oocyte cell-specific markers, and this expression increased in the third week of culture. In both groups of AP-MSCs and IP-MSCs, the expressions of Gdf9, Zp3, Ddx4, Dazl, and Amh genes under just HCCM induction showed upregulation significantly on the 21st day of culture compared with day 0. But in protein synthesis of all mentioned genes, both HFF and HCCM had equal induction effect on the 21st day of culture against the 0th day. In addition, LHR was not expressed in any groups. Finally, in both characterization and differentiation procedures, the AP-MSCs respond to inducers better than IP-MSCs.

Sialyllactose and Galactooligosaccharides Promote Epithelial Barrier Functioning and Distinctly Modulate Microbiota Composition and Short Chain Fatty Acid Production In Vitro.

Human milk oligosaccharides (HMO) and prebiotic oligosaccharides are proposed to confer several health benefits to the infant. They shape the microbiota, have anti-inflammatory properties, and support epithelial barrier functioning. However, in order to select the best oligosaccharides for inclusion in infant formulas, there is a need to increase our understanding of the specific effects of HMO and prebiotics on the host immune system. Therefore, we investigated the effects of the HMO sialyllactose (SL), and galactooligosaccharides (GOS) on epithelial barrier functioning, microbiota composition, and SCFA production. The effect of GOS and SL on epithelial barrier functioning and microbiota composition was investigated using in vitro models. Epithelial barrier function was investigated by transcriptome analysis of fully polarized Caco-2 cells exposed for 6 h to SL or GOS. In addition, epithelial cell growth, alkaline phosphatase production, and re-epithelization was studied. Further, we investigated the effect of SL and GOS on microbiota composition and SCFA production using in vitro fecal batch cultures. Transcriptome analysis showed that SL and GOS both induced pathways that regulate cell cycle control. This gene-expression profile translated to a phenotype of halted proliferation and included the induction of alkaline phosphatase activity, a marker of epithelial cell differentiation. SL and GOS also promoted re-epithelialization in an in vitro epithelial wound repair assay. SL and GOS did show distinct modulation of microbiota composition, promoting the outgrowth of Bacteroides and bifidobacteria, respectively, which resulted in distinct changes in SCFA production profiles. Our results show that SL and GOS can both modulate epithelial barrier function by inducing differentiation and epithelial wound repair, but differentially promote the growth of specific genera in the microbiota, which is associated with differential changes in SCFA profiles.

Establishment and characterization of an immortalized renal cell line of the Chinese tree shrew (Tupaia belangeri chinesis).

The Chinese tree shrew holds a great potential as a viable animal model in biomedical research, especially for infectious diseases and neuropsychiatric disorders. A thorough understanding of the innate immunity, which represents the first line that defends the host against viral infection, of the Chinese tree shrew, is needed. However, the progress is hindered by the lack of a proper cell line for research usage. In this study, we established a cell line that is applicable to the study of tree shrew innate immune responses against viral infections. The Chinese tree shrew primary renal cells (TSPRCs) were immortalized by simian virus 40 large T antigen (SV40LT) transduction, and the immortalized cells were termed TSR6 (tree shrew renal cell #6). TSR6 showed a similar morphology to TSPRCs and expressed the epithelial cell-specific marker cytokeratin 18 (KRT18). In addition, TSR6 could be transfected by transfection reagent and was suitable for CRISPR/Cas9-mediated gene editing. Infection of Newcastle disease virus (NDV) or herpes simplex virus 1 (HSV-1) in TSR6 induced the mRNA expression of tree shrew interferon-β (tIFNB1) and myxovirus resistance protein 1 (tMx1) in a dose- and time-dependent manner. Collectively, we successfully established a tree shrew renal cell line and demonstrated that this cell line was suitable for the study of the innate immune response to viral infections.