The role of L-carnitine in bovine embryo metabolism. A review of the effect of supplementation with a metabolic modulator on in vitro embryo production.

Early embryo development is driven first by the maternal RNAs and proteins accumulated during the oocyte's cytoplasmic maturation and then after the embryo genome activation. In mammalian cells, ATP generation occurs via oxidative pathways or by glycolysis, whereas in embryonic stem cells, the consumption of glucose, pyruvate, lipids, and amino acids results in ATP synthesis. Although the bovine embryo has energy reserves in glycogen and lipids, the glycogen concentration is deficient. Conversely, lipids represent the most abundant energy reservoir of bovine embryos, where lipid droplets-containing triacylglycerols are the main fatty acid stores. Oocytes of many mammalian species contain comparatively high amounts of lipids stored as droplets in the ooplasm. L-carnitine has been described as a cofactor that facilitates the mobilization of fatty acids present in the oocyte's cytoplasm into the mitochondria to facilitate ß-oxidation processes. However, the L-carnitine effects by addition to media in the in vitro produced embryos on the quality are highly disputed and contradictory by different researchers. This review's objective was to explore the effect that the addition of L-carnitine on culture media could have on the overall bovine embryo production in vitro, from the oocyte metabolism to the modulation of gene expression in the developing embryos.

Impaired Gene Expression Due to Iodine Excess in the Development and Differentiation of Endoderm and Thyroid Is Associated with Epigenetic Changes.

Background: Thyroid hormone (TH) synthesis is essential for the control of development, growth, and metabolism in vertebrates and depends on a sufficient dietary iodine intake. Importantly, both iodine deficiency and iodine excess (IE) impair TH synthesis, causing serious health problems especially during fetal/neonatal development. While it is known that IE disrupts thyroid function by inhibiting thyroid gene expression, its effects on thyroid development are less clear. Accordingly, this study sought to investigate the effects of IE during the embryonic development/differentiation of endoderm and the thyroid gland. Methods: We used the murine embryonic stem (ES) cell model of in vitro directed differentiation to assess the impact of IE on the generation of endoderm and thyroid cells. Additionally, we subjected endoderm and thyroid explants obtained during early gestation to IE and evaluated gene and protein expression of endodermal markers in both models. Results: ES cells were successfully differentiated into endoderm cells and, subsequently, into thyrocytes expressing the specific thyroid markers Tshr, Slc5a5, Tpo, and Tg. IE exposure decreased the messenger RNA (mRNA) levels of the main endoderm markers Afp, Crcx4, Foxa1, Foxa2, and Sox17 in both ES cell-derived endoderm cells and embryonic explants. Interestingly, IE also decreased the expression of the main thyroid markers in ES cell-derived thyrocytes and thyroid explants. Finally, we demonstrate that DNA methyltransferase expression was increased by exposure to IE, and this was accompanied by hypermethylation and hypoacetylation of histone H3, pointing to an association between the gene repression triggered by IE and the observed epigenetic changes. Conclusions: These data establish that IE treatment is deleterious for embryonic endoderm and thyroid gene expression.

FGF2 Stimulates the Growth and Improves the Melanocytic Commitment of Trunk Neural Crest Cells.

Neural crest cells (NCCs) comprise a population of multipotent progenitors and stem cells at the origin of the peripheral nervous system (PNS) and melanocytes of skin, which are profoundly influenced by microenvironmental factors, among which is basic fibroblast growth factor 2 (FGF2). In this work, we further investigated the role of this growth factor in quail trunk NC morphogenesis and demonstrated its huge effect in NCC growth mainly by stimulating cell proliferation but also reducing cell death, despite that NCC migration from the neural tube explant was not affected. Moreover, following FGF2 treatment, reduced expression of the early NC markers Sox10 and FoxD3 and improved proliferation of HNK1-positive NCC were observed. Since these markers are involved in the regulation of glial and melanocytic fate of NC, the effect of FGF2 on NCC differentiation was investigated. Therefore, in the presence of FGF2, increased proportions of NCCs positives to the melanoblast marker Mitf as well as NCCs double stained to Mitf and BrdU were recorded. In addition, treatment with FGF2, followed by differentiation medium, resulted in increased expression of melanin and improved proportion of melanin-pigmented melanocytes without alteration in the glial marker Schwann myelin protein (SMP). Taken together, these data further reveal the important role of FGF2 in NCC proliferation, survival, and differentiation, particularly in melanocyte development. This is the first demonstration of FGF2 effects in melanocyte commitment of NC and in the proliferation of Mitf-positive melanoblasts. Elucidating the differentiation process of embryonic NCCs brings us a step closer to understanding the development of the PNS and then undertaking the search for advanced technologies to prevent, or treat, injuries caused by NC-related disorders, also known as neurocristopathies.

PEG-PCL Nanocapsules Containing Curcumin: Validation of HPLC Method for Analyzing Drug-loading Efficiency, Stability Testing and Cytotoxicity on NIH-3T3 Cell Line

Abstract Curcumin (CUR) shows potential use for treating cancer. However, CUR has low solubility and reduced bioavailability, which limit its clinical effect. Therefore, the development of nanocarriers can overcome these problems and can ensure the desired pharmacological effect. In addition, it is mandatory to prove the quality, the efficacy, and the safety for a novel nanomedicine to be approved. In that sense, this paper aimed (a) to prepare CUR-loaded polyethylene glycol-poly(ε-caprolactone) nanocapsules; (b) to validate an analytical method by high performance liquid chromatography (HPLC) for quantifying CUR in these nanoformulations; (c) to evaluate the physicochemical stability of these formulations; and to investigate their cytotoxicity on NIH-3T3 mouse fibroblast cells. The HPLC method was specific to CUR in the loaded nanocapsules, linear (r = 0.9994) in a range of 10.0 to 90.0 µg.mL-1 with limits of detection and quantification of 0.160 and 0.480 µg.mL-1, respectively. Precision was demonstrated by a relative standard deviation lower than 5%. Suitable accuracy (102.37 ± 0.92%) was obtained. Values of pH, particle size, polydispersity index, and zeta potential presented no statistical difference (p > 0.05) for CUR-loaded nanoparticles. No cytotoxicity was observed against NIH-3T3 mouse embryo fibroblast cell line using both the tetrazolium salt and sulforhodamine B assays. In conclusion, a simple and inexpensive HPLC method was validated for the CUR quantification in the suspensions of nanocapsules. The obtained polymeric nanocapsules containing CUR showed suitable results for all the performed assays and can be further investigated as a feasible novel approach for cancer treatment.

Aluminum affects neural phenotype determination of embryonic neural progenitor cells.

Aluminum (Al) is a neurotoxin and is associated with the etiology of neurodegenerative diseases, such as Alzheimer's disease (AD). The Al-free ion (Al3+) is the biologically reactive and toxic form. However, the underlying mechanisms of Al toxicity in the brain remain unclear. Here, we evaluated the effects of Al3+ (in the chloride form-AlCl3) at different concentrations (0.1-100 µM) on the morphology, proliferation, apoptosis, migration and differentiation of neural progenitor cells (NPCs) isolated from embryonic telencephalons, cultured as neurospheres. Our results reveal that Al3+ at 100 µM reduced the number and diameter of neurospheres. Cell cycle analysis showed that Al3+ had a decisive function in proliferation inhibition of NPCs during neural differentiation and induced apoptosis on neurospheres. In addition, 1 µM Al3+ resulted in deleterious effects on neural phenotype determination. Flow cytometry and immunocytochemistry analysis showed that Al3+ promoted a decrease in immature neuronal marker ß3-tubulin expression and an increase in co-expression of the NPC marker nestin and glial fibrillary acidic protein. Thus, our findings indicate that Al3+ caused cellular damage and reduced proliferation and migration, resulting in global inhibition of NPC differentiation and neurogenesis.

Toxicity and in vivo release profile of sirolimus from implants into the vitreous of rabbits' eyes.

PURPOSE: To assess the in vivo release profile and the retinal toxicity of a poly (lactic-co-glycolic acid) (PLGA) sustained-release sirolimus (SRL) intravitreal implant in normal rabbit eyes. METHODS: PLGA intravitreal implants containing or not SRL were prepared, and the viability of ARPE-19 and hES-RPE human retinal cell lines was examined after 24 and 72 h of exposure to implants. New Zealand rabbits were randomly divided into two groups that received intravitreal implants containing or not SRL. At each time point (1-8 weeks), four animals from the SRL group were euthanized, the vitreous was collected, and drug concentration was calculated. Clinical evaluation of the eyes was performed weekly for 8 weeks after administration. Electroretinography (ERG) was recorded in other eight animals, four for each group, at baseline and at 24 h, 1, 4, 6, and 8 weeks after the injection. ERG was carried out using scotopic and photopic protocols. The safety of the implants was assessed using statistical analysis of the ERG parameters (a and b waves, a and b implicit time, B/A ratio, oscillatory potential, and Naka-Rushton analysis) comparing the functional integrity of the retina between the PLGA and SRL-PLGA groups. After the last electrophysiological assessment, the rabbits were euthanized and retinal histopathology was realized. RESULTS: After 24 and 72 h of incubation with PLGA or SRL-PLGA implants, ARPE-19 and hES-RPE cells showed viability over 70%. The maximum concentration of SRL (199.8 ng/mL) released from the device occurred within 4 weeks. No toxic effects of the implants or increase in the intraocular pressure was observed through clinical evaluation of the eye. ERG responses showed no significant difference between the eyes that received PLGA or SRL-PLGA implants at baseline and throughout the 8 weeks of follow-up. No remarkable difference in retinal histopathology was detected in rabbit eyes treated with PLGA or SRL-PLGA implants. CONCLUSIONS: Intravitreal PLGA or SRL-PLGA implants caused no significant reduction in cell viability and showed no evident toxic effect on the function or structure of the retina of the animals. SRL was released from PLGA implant after application in the vitreous of rabbits during 8 weeks.

Sex differences in depolarizing actions of GABAA receptor activation in rat embryonic hypothalamic neurons.

GABAA receptor activation exerts trophic actions in immature neurons through depolarization of resting membrane potential. The switch to its classical hyperpolarizing role is developmentally regulated. Previous results suggest that a hormonally biased sex difference exists at the onset of the switch in hypothalamic neurons. The aim of this work was to evaluate sex differences in GABAA receptor function of hypothalamic neurons before brain masculinization by gonadal hormones. Hypothalamic cells were obtained from embryonic day 16 male and female rat foetuses, 2 days before the peak of testosterone production by the foetal testis, and grown in vitro for 9 days. Whole-cell and perforated patch-clamp recordings were carried out in order to measure several electrophysiological parameters. Our results show that there are more male than female neurons responding with depolarization to muscimol. Additionally, among cells with depolarizing responses, males have higher and longer lasting responses than females. These results highlight the relevance of differences in neural cell sex irrespective of exposure to sex hormones.

Effects of glial cell line-derived neurotrophic factor and leukemia-inhibitory factor on the behavior of two calf testis germline stem cell colony types.

Germline stem cells are the only such capable of transmitting genetic information in vivo. The isolation and culture of these cells in vitro provide a unique model to understand sperm differentiation and hence, spermatogenesis and male fertility. In this study, we isolated, purified, and cultured germline stem cells from the testes of newborn calves. Moreover, we investigated the effects of glial cell line-derived neurotrophic factor (GDNF) and leukemia-inhibitory factor (LIF) on their proliferation. Male calf germline stem cells were found to be pluripotent, and able to form grape-like and embryonic stem cell (ES)-like colonies when cultured. GDNF promoted proliferation of the former, whereas LIF induced growth of the latter. The grape-like colonies retained their germline stem cell characteristics, whereas the ES-like colonies demonstrated more primitive attributes. This investigation established a male calf germline stem cell culture model that may serve as a foundation for further studies aiming to understand the properties of such cells.

Different concentrations of kaempferol distinctly modulate murine embryonic stem cell function.

Kaempferol (3,4',5,7-tetrahydroxyflavone) is a natural flavonoid with several beneficial and protective effects. It has been demonstrated that kaempferol has anticancer properties, particularly due to its effects on proliferation, apoptosis and the cell cycle. However, possible effects on pluripotent embryonic stem cell function have not yet been addressed. Embryonic stem cells have the ability to self-renew and to differentiate into all three germ layers with potential applications in regenerative medicine and in vitro toxicology. We show that exposure of murine embryonic stem cells (mESC) to high concentrations of kaempferol (200 µM) leads to decreased cell numbers, although the resulting smaller cell colonies remain pluripotent. However, lower concentrations of this compound (20 µM) increase the expression of pluripotency markers in mESCs. Mitochondrial membrane potential and mitochondrial mass are not affected, but a dose-dependent increase in apoptosis takes place. Moreover, mESC differentiation is impaired by kaempferol, which was not related to apoptosis induction. Our results show that low concentrations of kaempferol can be beneficial for pluripotency, but inhibit proper differentiation of mESCs. Additionally, high concentrations induce apoptosis and increase mitochondrial reactive oxygen species (ROS).

Estradiol differentially induces progesterone receptor isoforms expression through alternative promoter regulation in a mouse embryonic hypothalamic cell line.

Progesterone receptor (PR) presents two main isoforms (PR-A and PR-B) that are regulated by two specific promoters and transcribed from alternative transcriptional start sites. The molecular regulation of PR isoforms expression in embryonic hypothalamus is poorly understood. The aim of the present study was to assess estradiol regulation of PR isoforms in a mouse embryonic hypothalamic cell line (mHypoE-N42), as well as the transcriptional status of their promoters. MHypoE-N42 cells were treated with estradiol for 6 and 12 h. Then, Western blot, real-time quantitative reverse transcription polymerase chain reaction, and chromatin and DNA immunoprecipitation experiments were performed. PR-B expression was transiently induced by estradiol after 6 h of treatment in an estrogen receptor alpha (ERα)-dependent manner. This induction was associated with an increase in ERα phosphorylation (serine 118) and its recruitment to PR-B promoter. After 12 h of estradiol exposure, a downregulation of this PR isoform was associated with a decrease of specific protein 1, histone 3 lysine 4 trimethylation, and RNA polymerase II occupancy on PR-B promoter, without changes in DNA methylation and hydroxymethylation. In contrast, there were no estradiol-dependent changes in PR-A expression that could be related with the epigenetic marks or the transcription factors evaluated. We demonstrate that PR isoforms are differentially regulated by estradiol and that the induction of PR-B expression is associated to specific transcription factors interactions and epigenetic changes in its promoter in embryonic hypothalamic cells.

Propofol induces apoptosis and inhibits the proliferation of rat embryonic neural stem cells via gamma-aminobutyric acid type A receptor.

We investigated the effect of propofol on the proliferation and viability of rat embryonic neural stem cells (rENSCs) and the potential mechanisms involved. rENSCs were isolated and cultured in vitro and treated with 1, 10, or 50 µM propofol, while the control group was treated with 0.1 µM dimethyl sulfoxide. The effect of propofol on the proliferation and viability of rENSCs was examined by proliferation and apoptosis assays. Real-time polymerase chain reaction was employed to analyze the mRNA expression of checkpoint kinase 1 (Chk1) and p53 in rENSCs exposed to propofol. Immunoprecipitation assay and western blotting analysis were performed to analyze the effect of propofol on Chk1 and p53 activity. The gamma-aminobutyric acid type A (GABAA) receptor antagonist securinine was added to the rENSCs before being treated with propofol to investigate the role of the GABAA receptor in propofol-triggered effects on rENSCs. rENSCs specifically expressing nestin protein were successfully isolated and cultured for experiments. The inhibitory effect of propofol on rENSCs increased dose-dependently. The percentage of apoptotic cells increased to 11.7% and the activity of Chk1 and p53 enhanced after treatment with 50 µM propofol. However, addition of securinine abrogated propofol-induced apoptosis and activation of Chk1. The GABAA receptor mediates propofol-induced apoptosis and proliferation inhibition of rENSCs, possibly by modulating the Chk1/p53 signaling pathway.

Embryonic neural stem cells in a 3D bioassay for trophic stimulation studies.

Progenitors were discovered in the corpus striatum several years ago, but little is known about their proliferation and differentiation. The aim of this study was to analyze embryonic progenitor cells from the corpus striatum using a bioassay with trophic stimulation. Primary cells obtained from brains of rat embryos at E13-14 were dissected from striatum niches and cultured in stem cell media. These floating dispersed cells clumped together to forming floating bodies like irregular spheres (spheroids), which were placed in type I collagen gel and cultured under basal conditions or with the addition of NGF, NT-3, or NTN. Optimum growth of neurites was obtained, and after 24 and 48 h, they were measured for number and length. The expression of proliferation markers such as PCNA and Ki67, and of neural progenitor markers such as GFAP, nestin, vimentin, O4, A2B5, Pax6, S100, TubIII, and NeuN, was then analyzed. The initial behavior in cell cultures showed distinguishable spheroids that, when placed in 3D gels and with trophic support, generated neurites. A similar effect was observed in glial cell outgrowth from the spheroids. Our assay showed high reproducibility, short culture time, and high resolution for tracing neuron-neurite outgrowth or visualizing glial outgrowth in a few hours.

EDA-containing fibronectin increases proliferation of embryonic stem cells.

Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA(+)). Here, we investigated if the FN EDA(+) isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA(-)), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC's proliferation rate. Here we showed for the first time that this FN isoform enhances ESC's proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy.

2,4-Dinitrophenol induces neural differentiation of murine embryonic stem cells.

2,4-Dinitrophenol (DNP) is a neuroprotective compound previously shown to promote neuronal differentiation in a neuroblastoma cell line and neurite outgrowth in primary neurons. Here, we tested the hypothesis that DNP could induce neurogenesis in embryonic stem cells (ESCs). Murine ESCs, grown as embryoid bodies (EBs), were exposed to 20 µM DNP (or vehicle) for 4 days. Significant increases in the proportion of nestin- and ß-tubulin III-positive cells were detected after EB exposure to DNP, accompanied by enhanced glial fibrillary acidic protein (GFAP), phosphorylated extracellular signal-regulated kinase (p-ERK) and ATP-linked oxygen consumption, thought to mediate DNP-induced neural differentiation. DNP further protected ESCs from cell death, as indicated by reduced caspase-3 positive cells, and increased proliferation. Cell migration from EBs was significantly higher in DNP-treated EBs, and migrating cells were positive for nestin, ß-tubulin III and MAP2, similar to that observed with retinoic acid (RA)-treated EBs. Compared to RA, however, DNP exerted a marked neuritogenic effect on differentiating ESCs, increasing the average length and number of neurites per cell. Results establish that DNP induces neural differentiation of ESCs, accompanied by cell proliferation, migration and neuritogenesis, suggesting that DNP may be a novel tool to induce neurogenesis in embryonic stem cells.

Effect of antibiotics against Mycoplasma sp. on human embryonic stem cells undifferentiated status, pluripotency, cell viability and growth.

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate into specialized cells and hold great promise as models for human development and disease studies, cell-replacement therapies, drug discovery and in vitro cytotoxicity tests. The culture and differentiation of these cells are both complex and expensive, so it is essential to extreme aseptic conditions. hESCs are susceptible to Mycoplasma sp. infection, which is hard to detect and alters stem cell-associated properties. The purpose of this work was to evaluate the efficacy and cytotoxic effect of Plasmocin(TM) and ciprofloxacin (specific antibiotics used for Mycoplasma sp. eradication) on hESCs. Mycoplasma sp. infected HUES-5 884 (H5 884, stable hESCs H5-brachyury promoter-GFP line) cells were effectively cured with a 14 days Plasmocin(TM) 25 µg/ml treatment (curative treatment) while maintaining stemness characteristic features. Furthermore, cured H5 884 cells exhibit the same karyotype as the parental H5 line and expressed GFP, through up-regulation of brachyury promoter, at day 4 of differentiation onset. Moreover, H5 cells treated with ciprofloxacin 10 µg/ml for 14 days (mimic of curative treatment) and H5 and WA09 (H9) hESCs treated with Plasmocin(TM) 5 µg/ml (prophylactic treatment) for 5 passages retained hESCs features, as judged by the expression of stemness-related genes (TRA1-60, TRA1-81, SSEA-4, Oct-4, Nanog) at mRNA and protein levels. In addition, the presence of specific markers of the three germ layers (brachyury, Nkx2.5 and cTnT: mesoderm; AFP: endoderm; nestin and Pax-6: ectoderm) was verified in in vitro differentiated antibiotic-treated hESCs. In conclusion, we found that Plasmocin(TM) and ciprofloxacin do not affect hESCs stemness and pluripotency nor cell viability. However, curative treatments slightly diminished cell growth rate. This cytotoxic effect was reversible as cells regained normal growth rate upon antibiotic withdrawal.

Matrix-bound heparan sulfate is essential for the growth and pluripotency of human embryonic stem cells.

Human embryonic stem (hES) cell production of heparan sulfate influences cell fate and pluripotency. Human ES cells remain pluripotent in vitro through the action of growth factors signaling, and the activity of these factors depends on interaction with specific receptors and also with heparan sulfate. Here, we tested the hypothesis that matrix-associated heparan sulfate is enough to maintain hES cells under low fibroblast growth factor-2 concentration in the absence of live feeder cells. To pursue this goal, we compared hES cells cultured either on coated plates containing live murine embryonic fibroblasts (MEFs) or on a matrix derived from ethanol-fixed MEFs. hES cells were analyzed for the expression of pluripotency markers and the ability to form embryoid bodies. hES cells cultured either on live mouse fibroblasts or onto a matrix derived from fixed fibroblasts expressed similar levels of Oct-4, SOX-2, Nanog, TRA-1-60 and SSEA-4, and they were also able to form cavitated embryoid bodies. Heparan sulfate-depleted matrix lost the ability to support the adherence and growth of hES cells, confirming that this glycosaminoglycan, bound to the extracellular matrix, is enough for the growth and attachment of hES cells. Finally, we observed that the ethanol-fixed matrix decreases by 30% the levels of Neu5Gc in hES cells, indicating that this procedure reduces xeno-contamination. Our data suggest that matrix-bound heparan sulfate is required for the growth and pluripotency of hES cells and that ethanol-fixed MEFs may be used as a "live cell"-free substrate for stem cells.

Scaling-up of dental pulp stem cells isolated from multiple niches.

Dental pulp (DP) can be extracted from child's primary teeth (deciduous), whose loss occurs spontaneously by about 5 to 12 years. Thus, DP presents an easy accessible source of stem cells without ethical concerns. Substantial quantities of stem cells of an excellent quality and at early (2-5) passages are necessary for clinical use, which currently is a problem for use of adult stem cells. Herein, DPs were cultured generating stem cells at least during six months through multiple mechanical transfers into a new culture dish every 3-4 days. We compared stem cells isolated from the same DP before (early population, EP) and six months after several mechanical transfers (late population, LP). No changes, in both EP and LP, were observed in morphology, expression of stem cells markers (nestin, vimentin, fibronectin, SH2, SH3 and Oct3/4), chondrogenic and myogenic differentiation potential, even after cryopreservation. Six hours after DP extraction and in vitro plating, rare 5-bromo-2'-deoxyuridine (BrdU) positive cells were observed in pulp central part. After 72 hours, BrdU positive cells increased in number and were found in DP periphery, thus originating a multicellular population of stem cells of high purity. Multiple stem cell niches were identified in different zones of DP, because abundant expression of nestin, vimentin and Oct3/4 proteins was observed, while STRO-1 protein localization was restricted to perivascular niche. Our finding is of importance for the future of stem cell therapies, providing scaling-up of stem cells at early passages with minimum risk of losing their "stemness".

Cycle arrest and aneuploidy induced by zidovudine in murine embryonic stem cells.

Zidovudine (3'-azido-3'-deoxythymidine; AZT) is a nucleoside analogue widely used for the treatment of acquired immune deficiency syndrome (AIDS). Medical guidelines recommend the use of AZT by pregnant women in order to reduce risk of HIV vertical transmission. Although it is efficacious, little is known about the side effects of AZT on embryonic development. In this sense, we used murine embryonic stem (mES) cells as a model to investigate the consequences of AZT exposure for embryogenesis. Firstly, mES colonies were incubated with AZT (50 or 100 µM) and cell cycle profile was evaluated. While 27.7 ± 5.43% of untreated mES cells were in G2/M phase, this percentage raised to 45.96 ± 4.18% after AZT exposure (100 µM). To identify whether accumulation of cells in G2/M phase could be related to chromosome missegregation with consequent cell cycle arrest, aneuploidy rate was evaluated after AZT treatment. Untreated colonies presented 39.6 ± 8.4% of cells aneuploid, while after AZT 100 µM treatment, the proportion of aneuploid cells raised to 67.8 ± 3.4% with prevalence of chromosome loss. This event was accompanied by micronuclei formation as AZT 100 µM treated mES cells presented a 2-fold increase compared to untreated ones. These data suggest that AZT exerts genotoxic effects and increases chromosome instability at early stages of embryonic development.

Liver sinusoidal endothelial cells support the survival and undifferentiated growth of the CGR8 mouse embryonic stem cell line: possible role of leukemia inhibitory factor (LIF).

Murine embryonic stem cells (muESC) are maintained and expanded in vitro by culturing in the presence of leukemia inhibitory factor (LIF) or by coculturing on murine embryonic fibroblast (MEF). Previously we have shown that liver sinusoidal endothelial cells (LSEC) promote the survival, proliferation and differentiation of hematopoietic stem cells. In the present study we investigated whether LSEC might promote the survival and undifferentiated growth of muESC. For these purposes, muESC (CGR8 cell line) were cultured on LSEC monolayers (muESC/LSEC) or in the presence of conditioned medium from LSEC cultures (muESC/LSEC-CM), both in the absence of LIF. Microscopic observation showed the growth of undifferentiated ESC colonies in both muESC/LSEC or muESC/LSEC-CM cultures. A significant reduction in the growth of undifferentiated ESC colonies was observed when ESC were cultured in LSEC-CM previously incubated with anti-LIF. RT-PCR and Western blot analysis showed that LSEC constitutively express LIF at the mRNA and protein level. At different times of culture, muESC were harvested and analyzed for the expression of embryonic markers (SSEA-1 and Oct-4) and differentiation capacity. Flow cytometry analysis showed the presence of a higher percentage of muESC (>90%) expressing SSEA-1 in muESC/LSEC-CM, as compared with muESC/LSEC cocultures. muESC obtained from both types of cultures formed embryoid bodies in vitro, and form teratomas in testicles of mice. These results provide the first evidence that LSEC support the in vitro survival, self-renewal, undifferentiated growth and differentiation capacity of the muESC CGR8 cell line.

Cysteine proteases in differentiation of embryonic stem cells into neural cells.

Glycosylated mouse cystatin C (mCysC), an endogenous inhibitor of cysteine cathepsin proteases (CP), has been suggested as a cofactor of ß-FGF to induce the differentiation of mouse embryonic stem cells into neural progenitor cells (NPCs). To investigate the possible role of CP in neural differentiation, we treated embryoid bodies (EBs) with (i) E64, an inhibitor of papain-like CP and of calpains, (ii) an inhibitor of cathepsin L (iCatL), (iii) an inhibitor of calpains (iCalp), or (iv) cystatins, and their ability to differentiate into neural cells was assessed. We show that the inhibition of CP induces a significant increase in Pax6 expression in EBs, leading to an increase in the number of nestin-positive cells after 3 days. Fourteen days after E64 treatment, we observed increased numbers of ß-III-tubulin-positive cells, showing greater percentage of immature neurons, and this feature persisted up to 24 days. At this point, we encountered higher numbers of neurons with inward Na(+) current compared with untreated EBs. Further, we show that mCysC and iCatL, but not unglycosylated egg white cystatin or iCalp, increased the numbers of NPCs. In contrast to E64 and iCatL, mCysC did not inhibit CP in EBs and its neural-inducing activity required ß-FGF. We propose that the inhibition of CP induces the differentiation of mouse embryonic stem cells into NPCs and neurons through a mechanism that is distinct from CysC-induced neural differentiation.