Jarosław Czyż

Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells

Mouse embryonic stem (ES) cells differentiate into cells of all three primary germ layers including endodermal cells that produce insulin in vitro. We show that constitutive expression of Pax4 (Pax4+), and to a lesser extent Pdx1 (Pdx1+), affects the differentiation of ES cells and significantly promote the development of insulin-producing cells. In Pax4 overexpressing R1 ES cells, isl-1, ngn3, insulin, islet amyloid polypeptide, and glucose transporter 2 (Glut-2) mRNA levels increase significantly. The number of nestin-expressing (nestin+) cells also increases. Constitutive Pax4 expression combined with selection of nestin+ cells and histotypic culture conditions give rise to spheroids containing insulin-positive granules typical of embryonal and adult β cells. In response to glucose, Pax4+ and wild-type ES-derived cells release insulin. Transplantation of these cells into streptozotocin-treated diabetic mice results in a normalization of blood glucose levels. We conclude that constitutive expression of Pax4 in combination with histotypic cultivation facilitates ES cell differentiation into the pancreatic lineage, which leads to the formation of islet-like spheroid structures that produce increased levels of insulin.

Differentiation of embryonic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors.

Here, we describe the generation of viable and dopamine-producing neurons derived from pluripotent mouse embryonic stem cells. Neurotrophic factors in combination with survival-promoting factors, such as interleukin-1beta, glial cell line-derived neurotrophic factor, neurturin, transforming growth factor-beta(3) and dibutyryl-cyclic AMP, significantly enhanced Nurr1 and tyrosine hydroxylase (TH) mRNA levels, whereas En-1, mash-1 and dopamine-2-receptor mRNA levels were not upregulated. In parallel, mRNA levels of the anti-apoptotic gene bcl-2 were found to be upregulated at terminal stages. Double immunofluorescence analysis revealed increased numbers of TH- and dopamine transporter-, but not gamma-aminobutyric acid- and serotonin-positive neurons in relation to synaptophysin-labeled cells by survival-promoting factors. Moreover, high-performance liquid chromatography analysis showed detectable levels of intracellular dopamine. We conclude that survival-promoting factors enhance differentiation, survival and maintenance of dopaminergic neurons derived from embryonic stem cells.

Electromagnetic fields affect transcript levels of apoptosis-related genes in embryonic stem cell-derived neural progenitor cells

Mouse embryonic stem (ES) cells were used as an experimental model to study the effects of electromagnetic fields (EMF). ES‐derived nestin‐positive neural progenitor cells were exposed to extremely low frequency EMF simulating power line magnetic fields at 50 Hz (ELF‐EMF) and to radiofrequency EMF simulating the Global System for Mobile Communication (GSM) signals at 1.71 GHz (RF‐EMF). Following EMF exposure, cells were analyzed for transcript levels of cell cycle regulatory, apoptosis‐related, and neural‐specific genes and proteins; changes in proliferation; apoptosis; and cytogenetic effects. Quantitative RT‐PCR analysis revealed that ELF‐EMF exposure to ES‐derived neural cells significantly affected transcript levels of the apoptosis‐related bcl‐2, bax, and cell cycle regulatory “growth arrest DNA damage inducible” GADD45 genes, whereas mRNA levels of neural‐specific genes were not affected. RF‐EMF exposure of neural progenitor cells resulted in down‐regulation of neural‐specific Nurr1 and in up‐regulation of bax and GADD45 mRNA levels. Short‐term RF‐EMF exposure for 6 h, but not for 48 h, resulted in a low and transient increase of DNA double‐strand breaks. No effects of ELF‐ and RF‐EMF on mitochondrial function, nuclear apoptosis, cell proliferation, and chromosomal alterations were observed. We may conclude that EMF exposure of ES‐derived neural progenitor cells transiently affects the transcript level of genes related to apoptosis and cell cycle control. However, these responses are not associated with detectable changes of cell physiology, suggesting compensatory mechanisms at the translational and posttranslational level.

Potential of embryonic and adult stem cells in vitro

Abstract Recent developments in the field of stem cell research indicate their enormous potential as a source of tissue for regenerative therapies. The success of such applications will depend on the precise properties and potentials of stem cells isolated either from embryonic, fetal or adult tissues. Embryonic stem cells established from the inner cell mass of early mouse embryos are characterized by nearly unlimited proliferation, and the capacity to differentiate into derivatives of essentially all lineages. The recent isolation and culture of human embryonic stem cell lines presents new opportunities for reconstructive medicine. However, important problems remain; first, the derivation of human embryonic stem cells from in vitro fertilized blastocysts creates ethical problems, and second, the current techniques for the directed differentiation into somatic cell populations yield impure products with tumorigenic potential. Recent studies have also suggested an unexpectedly wide developmental potential of adult tissuespecific stem cells. Here too, many questions remain concerning the nature and status of adult stem cells both in vivo and in vitro and their proliferation and differentiation/ transdifferentiation capacity. This review focuses on those issues of embryonic and adult stem cell biology most relevant to their in vitro propagation and differentiation. Questions and problems related to the use of human embryonic and adult stem cells in tissue regeneration and transplantation are discussed.

Antioxidant and anticancer activities of Chenopodium quinoa leaves extracts – In vitro study

The nutraceutical potential of Chenopodium quinoa Leaves (ChL) was assessed through analyses of their phenolic content, elucidation of the effect of ChL phenolic compounds on cancer cell properties and estimation of their antioxidative activity, bioaccessibility and bioavailability in vitro. Considerable amounts of ferulic, sinapinic and gallic acids, kaempferol, isorhamnetin and rutin were observed in the chemical ChL extract and were linked with its inhibitory effect on prostate cancer cell proliferation, motility and cellular competence for gap junctional communication. Both extracts, chemical and obtained after simulated digestion, exerted an inhibitory effect on lipoxygenase activity, paralleled by their considerable chelating, antioxidative, antiradical and reducing power. These observations indicate that phenolic ChL compounds may exert a chemopreventive and anticarcinogenic effect on oxidative stress and ROS-dependent intracellular signaling via synergic effects. The relatively high potential bioaccessibility and bioavailability of the compounds probably responsible for these effects demonstrates the suitability of ChL for dietary supplementation.

Flavonoid apigenin inhibits motility and invasiveness of carcinoma cells in vitro

Investigations of the mechanisms of the cancer‐preventive activity of apigenin (4′,5,7,‐trihydroxyflavone), a plant‐derived, anti‐carcinogenic flavonoid, showed its interference with cell proliferation, survival, and gap junctional coupling. We used a model based on non‐invasive HeLa wild‐type cells and their connexin43 (Cx43) transfected counterparts to correlate the effect of apigenin on tumour cell invasiveness with its influence on cell motility. Both cell lines displayed similar motile properties in control conditions. Apigenin treatment resulted in a significant and reversible inhibition of translocation of both HeLa wild‐type cells and HeLa Cx43 transfectants. The effect of apigenin on cell proliferation was less pronounced especially at low apigenin concentration, whereas its influence on cell motility correlated with the reduction of the invasive potential of HeLa Cx43 cells as shown by an invasion assay based on the confrontation of tumour cell spheroids with chick embryo heart fragments. HeLa Cx43 cells were highly invasive in controls, but did not invade the heart tissue at tumour cell aggregate‐fibroblast capsule interfaces in the presence of apigenin and failed to fully engulf these heart fragments. Because the motility of chick heart fibroblasts was only slightly affected by apigenin, these observations indicate that apigenin exerts its anti‐invasive effect on HeLa cells predominantly via a specific inhibition of tumour cell motility. This inhibitory effect of apigenin on tumour cell invasiveness in vitro demonstrates that apigenin may exert its anti‐tumorigenic effect in vivo via inhibition of tumour cell penetration of the healthy tissue.

The influence of protein–flavonoid interactions on protein digestibility in vitro and the antioxidant quality of breads enriched with onion skin

Different types of breads enriched with onion skin were studied. The objectives were twofold: to show and examine protein-phenolic interactions and to discuss results concerning phenolic content, antioxidant activity and protein digestibility in the light of in vitro bioaccessibility. Phenolic contents and antiradical abilities were linked with the level of onion skin supplement however, the amounts determined were significantly lower than expected. Fortification influenced protein digestibility (a reduction from 78.4% for control breads to 55% for breads with a 4% supplement). Electrophoretic and chromatographic studies showed the presence of indigestible protein-flavonoid complexes - with molecular weights about 25 kDa and 14.5 kDa; however, the reduction of free amino group levels and the increase in chromatogram areas suggest that flavonoids also bind to other bread proteins. The interaction of phenolics with proteins affects antioxidant efficacy and protein digestibility; thus, they have multiple effects on food quality and pro-health properties.

The stage-specific function of gap junctions during tumourigenesis.

Tumour development is a process resulting from the disturbance of various cellular functions including cell proliferation, adhesion and motility. While the role of these cell parameters in tumour promotion and progression has been widely recognized, the mechanisms that influence gap junctional coupling during tumorigenesis remain elusive. Neoplastic cells usually display decreased levels of connexin expression and/or gap junctional coupling. Thus, impaired intercellular communication via gap junctions may facilitate the release of a potentially neoplastic cell from the controlling regime of the surrounding tissue, leading to tumour promotion. However, recent data indicates that metastatic tumour cell lines are often characterized by relatively high levels of connexin expression and gap junctional coupling. This review outlines current knowledge on the role of connexins in tumorigenesis and the possible mechanisms of the interference of gap junctional coupling with the processes of tumour invasion and metastasis.

Quality and antioxidant properties of breads enriched with dry onion (Allium cepa L.) skin

The aim of the study was to investigate the effect on the antioxidant properties and sensory value of bread of adding ground onion skin (OS). For a determination of bioaccessibility and bioavailability in vitro the human gastrointestinal tract model was used. OS contained mastication-extractable quercetin (4.6 mg/g). Quercetin from OS was highly bioaccessible during in vitro conditions, but only approximately 4% of quercetin released during simulated digestion was bioavailable in vitro. The antioxidant potential of bread with OS was significantly higher than the activity noted in the control. In particular, OS addition significantly fortificated bread with bioaccessible lipid oxidation preventers and compounds with reducing and chelating abilities. The 2-3% OS addition caused significant improvement of antioxidant abilities (further increases in the OS supplement did not increase the activity of bread). Sensory evaluation showed that replacement of wheat flour in bread with up to 3% OS powder gave satisfactory consumer acceptability.

Contact stimulation of prostate cancer cell migration : the role of gap junctional coupling and migration stimulated by heterotypic cell-to-cell contacts in determination of the metastatic phenotype of Dunning rat prostate cancer cells

Background information. Motile activity of tumour cells is regarded as a critical factor determining their metastatic potential. We have shown previously that contrary to the majority of normal cells, homotypic contacts between some tumour cells, among them low metastatic (AT‐2) and highly metastatic (MAT‐LyLu) rat prostate cancer cells, increase the speed of their movements. The aim of the present study was to determine the effect of heterotypic cell‐to‐cell contacts on the migration of rat prostate cancer cells differing in metastatic potential, and to correlate it with the intensity of homo‐ and heterologous gap junctional coupling.