In Vitro Germ Cell Differentiation from Cynomolgus Monkey Embryonic Stem Cells

Background

Mouse embryonic stem (ES) cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro.

Methods and Findings

To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis). VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB) formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA) 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene.

Conclusion

VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the identification and characterization of germ cells derived from ES cells are possible by using reported germ cell markers in vivo, including SSEA1, OCT-4, and VASA, in vitro as well as in vivo. These findings are thus considered to help elucidate the germ cell developmental process in primates.     

Mechanisms by Which Low Glucose Enhances the Cytotoxicity of Metformin to Cancer Cells Both In Vitro and In Vivo

Different cancer cells exhibit altered sensitivity to metformin treatment. Recent studies suggest these findings may be due in part to the common cell culture practice of utilizing high glucose, and when glucose is lowered, metformin becomes increasingly cytotoxic to cancer cells. In low glucose conditions ranging from 0 to 5 mM, metformin was cytotoxic to breast cancer cell lines MCF7, MDAMB231 and SKBR3, and ovarian cancer cell lines OVCAR3, and PA-1. MDAMB231 and SKBR3 were previously shown to be resistant to metformin in normal high glucose medium. When glucose was increased to 10 mM or above, all of these cell lines become less responsive to metformin treatment. Metformin treatment significantly reduced ATP levels in cells incubated in media with low glucose (2.5 mM), high fructose (25 mM) or galactose (25 mM). Reductions in ATP levels were not observed with high glucose (25 mM). This was compensated by enhanced glycolysis through activation of AMPK when oxidative phosphorylation was inhibited by metformin. However, enhanced glycolysis was either diminished or abolished by replacing 25 mM glucose with 2.5 mM glucose, 25 mM fructose or 25 mM galactose. These findings suggest that lowering glucose potentiates metformin induced cell death by reducing metformin stimulated glycolysis. Additionally, under low glucose conditions metformin significantly decreased phosphorylation of AKT and various targets of mTOR, while phospho-AMPK was not significantly altered. Thus inhibition of mTOR signaling appears to be independent of AMPK activation. Further in vivo studies using the 4T1 breast cancer mouse model confirmed that metformin inhibition of tumor growth was enhanced when serum glucose levels were reduced via low carbohydrate ketogenic diets. The data support a model in which metformin treatment of cancer cells in low glucose medium leads to cell death by decreasing ATP production and inhibition of survival signaling pathways. The enhanced cytotoxicity of metformin against cancer cells was observed both in vitro and in vivo.     

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells¹. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of ''cellular microbiology''². Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories^3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 2787⁵, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787ΔaidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before⁶.Download video file.^{(34M, mov)}     

In Vitro Cultivation of the Hymenoptera Genetic Model,Nasonia

The wasp genus Nasonia is a genetic model with unique advantages for the study of interspecific differences, including haplodiploidy and interfertile species. However, as a parasitoid, Nasonia is confined within a fly host, thus restricting direct observations and manipulation of development over time. Here, we present the first in vitro cultivation method for this system that decouples Nasonia from its host, allowing continuous observations from embryo to adulthood. Using transwell plates and a simple Nasonia rearing medium, we demonstrate a technique that will significantly expand the utility of the Nasonia model.     

In Vitro Characterization of Circulating Endothelial Progenitor Cells Isolated from Patients with Acute Coronary Syndrome

Background

The current understanding of the functional characteristics of circulating endothelial progenitor cells (EPC) is limited, especially in patients affected by cardiovascular diseases. In this study, we have analyzed the in vitro clonogenic capacity of circulating EPC, also known as endothelial colony-forming cells (ECFC), in patients with acute coronary syndrome (ACS), in comparison to the colony forming unit-endothelial-like cells (CFU-EC) of hematopoietic/monocytic origin.

Methodology/Principal Findings

By culturing peripheral blood mononuclear cells (PBMC) of patients with ACS (n = 70), CFU-EC were frequently isolated (from 77% of ACS patients), while EPC/ECFC were obtained only in a small subset (13%) of PBMC samples, all harvested between 7–14 days after the acute cardiovascular event. Notably, ex-vivo generation of EPC/ECFC was correlated to a higher in vitro release of PDGF-AA by the corresponding ACS patient PBMC. By using specific endothelial culture media, EPC/ECFC displayed in vitro expansion capacity, allowing the phenotypic and functional characterization of the cells. Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: i) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, i) variable expression of the CD34, CD106 and CD184 markers, and iii) negativity for CD45, CD90, CD117 and CD133. Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities.

Conclusion/Significance

Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.     

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells

In this study we used differentiated adult human upcyte^® cells for the in vitro generation of liver organoids. Upcyte^® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte^® process). Proliferating upcyte^® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte^® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.     

In Vitro Model of Tumor Cell Extravasation

Tumor cells that disseminate from the primary tumor and survive the vascular system can eventually extravasate across the endothelium to metastasize at a secondary site. In this study, we developed a microfluidic system to mimic tumor cell extravasation where cancer cells can transmigrate across an endothelial monolayer into a hydrogel that models the extracellular space. The experimental protocol is optimized to ensure the formation of an intact endothelium prior to the introduction of tumor cells and also to observe tumor cell extravasation by having a suitable tumor seeding density. Extravasation is observed for 38.8% of the tumor cells in contact with the endothelium within 1 day after their introduction. Permeability of the EC monolayer as measured by the diffusion of fluorescently-labeled dextran across the monolayer increased 3.8 fold 24 hours after introducing tumor cells, suggesting that the presence of tumor cells increases endothelial permeability. The percent of tumor cells extravasated remained nearly constant from1 to 3 days after tumor seeding, indicating extravasation in our system generally occurs within the first 24 hours of tumor cell contact with the endothelium.     

In Vitro and Sensory Evaluation of Capsaicin-Loaded Nanoformulations

Capsaicin has known health beneficial and therapeutic properties. It is also able to enhance the permeability of drugs across epithelial tissues. Unfortunately, due to its pungency the oral administration of capsaicin is limited. To this end, we assessed the effect of nanoencapsulation of capsaicin, under the hypothesis that this would reduce its pungency. Core-shell nanocapsules with an oily core and stabilized with phospholipids were used. This system was used with or without chitosan coating. In this work, we investigated the in vitro release behavior of capsaicin-loaded formulations in different physiological media (including simulated saliva fluid). We also evaluated the influence of encapsulation of capsaicin on the cell viability of buccal cells (TR146). To study the changes in pungency after encapsulation we carried out a sensory analysis with a trained panel of 24 students. The in vitro release study showed that the systems discharged capsaicin slowly in a monotonic manner and that the chitosan coating had an effect on the release profile. The cytotoxic response of TR146 cells to capsaicin at a concentration of 500 μM, which was evident for the free compound, was reduced following its encapsulation. The sensory study revealed that a chitosan coating results in a lower threshold of perception of the formulation. The nanoencapsulation of capsaicin resulted in attenuation of the sensation of pungency significantly. However, the presence of a chitosan shell around the nanoformulations did not mask the pungency, when compared with uncoated systems.     

GMP-Compliant,Large-Scale Expanded Allogeneic Natural Killer Cells Have Potent Cytolytic Activity against Cancer Cells In Vitro and In Vivo

Ex vivo-expanded, allogeneic natural killer (NK) cells can be used for the treatment of various types of cancer. In allogeneic NK cell therapy, NK cells from healthy donors must be expanded in order to obtain a sufficient number of highly purified, activated NK cells. In the present study, we established a simplified and efficient method for the large-scale expansion and activation of NK cells from healthy donors under good manufacturing practice (GMP) conditions. After a single step of magnetic depletion of CD3⁺ T cells, the depleted peripheral blood mononuclear cells (PBMCs) were stimulated and expanded with irradiated autologous PBMCs in the presence of OKT3 and IL-2 for 14 days, resulting in a highly pure population of CD3⁻CD16⁺CD56⁺ NK cells which is desired for allogeneic purpose. Compared with freshly isolated NK cells, these expanded NK cells showed robust cytokine production and potent cytolytic activity against various cancer cell lines. Of note, expanded NK cells selectively killed cancer cells without demonstrating cytotoxicity against allogeneic non-tumor cells in coculture assays. The anti-tumor activity of expanded human NK cells was examined in SCID mice injected with human lymphoma cells. In this model, expanded NK cells efficiently controlled lymphoma progression. In conclusion, allogeneic NK cells were efficiently expanded in a GMP-compliant facility and demonstrated potent anti-tumor activity both in vitro and in vivo.     

Effect of Some Antibiotics on the In Vitro Morphogenetic Response from Callus Cultures of Coryphantha Elephantidens

The effect of five antibiotics: carbenicillin, chloramphenicol, cefotaxime, kanamycin and hygromycin on the organogenesis from callus cultures of Coryphantha elphantidens (Lem.) Lem. have been studied. Carbenicillin and cefotaxime stimulated shoot regeneration from callus. All antibiotics under study suppressed rooting of in vitro formed shoots. After five sequential subcultures on kanamycin supplemented medium, antibiotic resistant callus was obtained. To study the impact of kanamycin on resistant callus, total protein content was also studied. Selected callus showed a remarkable increase in callus mass. Antibiotic resistant plants have been selected by screening callus pieces on kanamycin supplemented media. Total protein content increased with subsequent subcultures in kanamycin resistant callus. The kanamycin selected shoots withstood the stability test after 2 months on antibiotic free medium. Plants were raised from the callus, which formed roots in 20 mg dm^–3 kanamycin, which was under study.     

Cryptoporus volvatus Extract Inhibits Influenza Virus Replication In Vitro and In Vivo

Influenza virus is the cause of significant morbidity and mortality, posing a serious health threat worldwide. Here, we evaluated the antiviral activities of Cryptoporus volvatus extract on influenza virus infection. Our results demonstrated that the Cryptoporus volvatus extract inhibited different influenza virus strain replication in MDCK cells. Time course analysis indicated that the extract exerted its inhibition at earlier and late stages in the replication cycle of influenza virus. Subsequently, we confirmed that the extract suppressed virus internalization into and released from cells. Moreover, the extract significantly reduced H1N1/09 influenza virus load in lungs and dramatically decreased lung lesions in mice. And most importantly, the extract protected mice from lethal challenge with H1N1/09 influenza virus. Our results suggest that the Cryptoporus volvatus extract could be a potential candidate for the development of a new anti-influenza virus therapy.     

In Vitro and In Vivo Human Herpesvirus 8 Infection of Placenta

Herpesvirus infection of placenta may be harmful in pregnancy leading to disorders in fetal growth, premature delivery, miscarriage, or major congenital abnormalities. Although a correlation between human herpesvirus 8 (HHV-8) infection and abortion or low birth weight in children has been suggested, and rare cases of in utero or perinatal HHV-8 transmission have been documented, no direct evidence of HHV-8 infection of placenta has yet been reported. The aim of this study was to evaluate the in vitro and in vivo susceptibility of placental cells to HHV-8 infection. Short-term infection assays were performed on placental chorionic villi isolated from term placentae. Qualitative and quantitative HHV-8 detection were performed by PCR and real-time PCR, and HHV-8 proteins were analyzed by immunohistochemistry. Term placenta samples from HHV-8-seropositive women were analyzed for the presence of HHV-8 DNA and antigens. In vitro infected histocultures showed increasing amounts of HHV-8 DNA in tissues and supernatants; cyto- and syncitiotrophoblasts, as well as endothelial cells, expressed latent and lytic viral antigens. Increased apoptotic phenomena were visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end-labeling method in infected histocultures. Ex vivo, HHV-8 DNA and a latent viral antigen were detected in placenta samples from HHV-8-seropositive women. These findings demonstrate that HHV-8, like other human herpesviruses, may infect placental cells in vitro and in vivo, thus providing evidence that this phenomenon might influence vertical transmission and pregnancy outcome in HHV-8-infected women.     

Adult Stromal Cells Derived from Human Adipose Tissue Provoke Pancreatic Cancer Cell Death both In Vitro and In Vivo

Background

Normal tissue homeostasis is maintained by dynamic interactions between epithelial cells and their microenvironment. Disrupting this homeostasis can induce aberrant cell proliferation, adhesion, function and migration that might promote malignant behavior. Indeed, aberrant stromal-epithelial interactions contribute to pancreatic ductal adenocarcinoma (PDAC) spread and metastasis, and this raises the possibility that novel stroma-targeted therapies represent additional approaches for combating this malignant disease. The aim of the present study was to determine the effect of human stromal cells derived from adipose tissue (ADSC) on pancreatic tumor cell proliferation.

Principal Findings

Co-culturing pancreatic tumor cells with ADSC and ADSC-conditioned medium sampled from different donors inhibited cancer cell viability and proliferation. ADSC-mediated inhibitory effect was further extended to other epithelial cancer-derived cell lines (liver, colon, prostate). ADSC conditioned medium induced cancer cell necrosis following G1-phase arrest, without evidence of apoptosis. In vivo, a single intra-tumoral injection of ADSC in a model of pancreatic adenocarcinoma induced a strong and long-lasting inhibition of tumor growth.

Conclusion

These data indicate that ADSC strongly inhibit PDAC proliferation, both in vitro and in vivo and induce tumor cell death by altering cell cycle progression. Therefore, ADSC may constitute a potential cell-based therapeutic alternative for the treatment of PDAC for which no effective cure is available.     

In Vitro Identification of Histatin 5 Salivary Complexes

With recent progress in the analysis of the salivary proteome, the number of salivary proteins identified has increased dramatically. However, the physiological functions of many of the newly discovered proteins remain unclear. Closely related to the study of a protein’s function is the identification of its interaction partners. Although in saliva some proteins may act primarily as single monomeric units, a significant percentage of all salivary proteins, if not the majority, appear to act in complexes with partners to execute their diverse functions. Coimmunoprecipitation (Co-IP) and pull-down assays were used to identify the heterotypic complexes between histatin 5, a potent natural antifungal protein, and other salivary proteins in saliva. Classical protein–protein interaction methods in combination with high-throughput mass spectrometric techniques were carried out. Co-IP using protein G magnetic Sepharose TM beads suspension was able to capture salivary complexes formed between histatin 5 and its salivary protein partners. Pull-down assay was used to confirm histatin 5 protein partners. A total of 52 different proteins were identified to interact with histatin 5. The present study used proteomic approaches in conjunction with classical biochemical methods to investigate protein–protein interaction in human saliva. Our study demonstrated that when histatin 5 is complexed with salivary amylase, one of the 52 proteins identified as a histatin 5 partner, the antifungal activity of histatin 5 is reduced. We expected that our proteomic approach could serve as a basis for future studies on the mechanism and structural-characterization of those salivary protein interactions to understand their clinical significance.     

Streptococcus mitis Induces Conversion of Helicobacter pylori to Coccoid Cells during Co-Culture In Vitro

Helicobacter pylori (H. pylori) is a major gastric pathogen that has been associated with humans for more than 60,000 years. H. pylori causes different gastric diseases including dyspepsia, ulcers and gastric cancers. Disease development depends on several factors including the infecting H. pylori strain, environmental and host factors. Another factor that might influence H. pylori colonization and diseases is the gastric microbiota that was overlooked for long because of the belief that human stomach was a hostile environment that cannot support microbial life. Once established, H. pylori mainly resides in the gastric mucosa and interacts with the resident bacteria. How these interactions impact on H. pylori-caused diseases has been poorly studied in human. In this study, we analyzed the interactions between H. pylori and two bacteria, Streptocccus mitis and Lactobacillus fermentum that are present in the stomach of both healthy and gastric disease human patients. We have found that S. mitis produced and released one or more diffusible factors that induce growth inhibition and coccoid conversion of H. pylori cells. In contrast, both H. pylori and L. fermentum secreted factors that promote survival of S. mitis during the stationary phase of growth. Using a metabolomics approach, we identified compounds that might be responsible for the conversion of H. pylori from spiral to coccoid cells. This study provide evidences that gastric bacteria influences H. pylori physiology and therefore possibly the diseases this bacterium causes.     

In Vitro Cultivation of Borrelia duttonii on Cultures of SflEp Cells

Borrelia duttonii strain 406 K, a causative agent of relapsing fever, could not be cultivated in vitro in currently available media for borreliae. We have developed an in vitro cultivation system by using SflEp cell cultures. The average increases of the number of borreliae, when inoculated with 1.0 × 10⁵ organisms per ml from infected mice, were 23-fold and 150-fold in the primary culture and the 3rd subculture, respectively. Even a single borrelia could propagate in this cultivation system. This system will be useful for immunological and physiological studies on uncultivable Borrelia strains.     

Cyclopia Extracts Act as ERα Antagonists and ERβ Agonists,In Vitro and In Vivo

Hormone replacement therapy associated risks, and the concomitant reluctance of usage, has instigated the search for new generations of estrogen analogues that would maintain estrogen benefits without associated risks. Furthermore, if these analogues display chemo-preventative properties in breast and endometrial tissues it would be of great value. Both the selective estrogen receptor modulators as well as the selective estrogen receptor subtype modulators have been proposed as estrogen analogues with improved risk profiles. Phytoestrogen containing extracts of Cyclopia, an indigenous South African fynbos plant used to prepare Honeybush tea may serve as a source of new estrogen analogues. In this study three extracts, P104, SM6Met, and cup-of-tea, from two species of Cyclopia, C. genistoides and C. subternata, were evaluated for ER subtype specific agonism and antagonism both in transactivation and transrepression. For transactivation, the Cyclopia extracts displayed ERα antagonism and ERβ agonism when ER subtypes were expressed separately, however, when co-expressed only agonism was uniformly observed. In contrast, for transrepression, this uniform behavior was lost, with some extracts (P104) displaying uniform agonism, while others (SM6Met) displayed antagonism when subtypes were expressed separately and agonism when co-expressed. In addition, breast cancer cell proliferation assays indicate that extracts antagonize cell proliferation in the presence of estrogen at lower concentrations than that required for proliferation. Furthermore, lack of uterine growth and delayed vaginal opening in an immature rat uterotrophic model validates the ERα antagonism of extracts observed in vitro and supports the potential of the Cyclopia extracts as a source of estrogen analogues with a reduced risk profile.     

In Vitro,In Silico and In Vivo Studies of Ursolic Acid as an Anti-Filarial Agent

As part of our drug discovery program for anti-filarial agents from Indian medicinal plants, leaves of Eucalyptus tereticornis were chemically investigated, which resulted in the isolation and characterization of an anti-filarial agent, ursolic acid (UA) as a major constituent. Antifilarial activity of UA against the human lymphatic filarial parasite Brugia malayi using in vitro and in vivo assays, and in silico docking search on glutathione-s-transferase (GST) parasitic enzyme were carried out. The UA was lethal to microfilariae (mf; LC₁₀₀: 50; IC₅₀: 8.84 µM) and female adult worms (LC₁₀₀: 100; IC50: 35.36 µM) as observed by motility assay; it exerted 86% inhibition in MTT reduction potential of the adult parasites. The selectivity index (SI) of UA for the parasites was found safe. This was supported by the molecular docking studies, which showed adequate docking (LibDock) scores for UA (−8.6) with respect to the standard antifilarial drugs, ivermectin (IVM −8.4) and diethylcarbamazine (DEC-C −4.6) on glutathione-s-transferase enzyme. Further, in silico pharmacokinetic and drug-likeness studies showed that UA possesses drug-like properties. Furthermore, UA was evaluated in vivo in B. malayi-M. coucha model (natural infection), which showed 54% macrofilaricidal activity, 56% female worm sterility and almost unchanged microfilaraemia maintained throughout observation period with no adverse effect on the host. Thus, in conclusion in vitro, in silico and in vivo results indicate that UA is a promising, inexpensive, widely available natural lead, which can be designed and developed into a macrofilaricidal drug. To the best of our knowledge this is the first ever report on the anti-filarial potential of UA from E. tereticornis, which is in full agreement with the Thomson Reuter''s ‘Metadrug’ tool screening predictions.