A Low Ethanol Dose Affects all Types of Cells in Mixed Long-Term Embryonic Cultures of the Cerebellum

DEFF Research Database (Denmark)

Pickering, Chris; Wicher, Grzegorz; Rosendahl, Sofi

2010-01-01

of this ethanol dose, cultures were exposed for 30 days. After this period, virtually no neurons or myelinating oligodendrocytes were present in the ethanol-treated cultures. In conclusion, chronic exposure to ethanol, even at small doses, dramatically and persistently affects normal development........ We exposed a primary culture of rat cerebellum from embryonic day 17 (corresponding to second trimester in humans) to ethanol at a concentration of 17.6 mM which is roughly equivalent to one glass of wine. Acutely, there was no change in cell viability after 5 or 8 days of exposure relative...... to control. By 11 days, a reduction in the number of viable cells was observed without an accompanying change in caspase-3 activity (marker of apoptotic cell death), suggesting changes in cell proliferation. As the proportion of nestin-positive cells was higher in the ethanol-treated cultures after 5 days...

Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

DEFF Research Database (Denmark)

Meyer, Morten; Widmer, H R; Wagner, B

1998-01-01

of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral...... numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 +/- 98, mean +/- SEM) and grafts of fresh, dissociated cell suspension (806 +/- 105, mean +/- SEM). Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after......Ventral mesencephalon (VM) of fetal rat and human origin grown as free-floating roller-tube (FFRT) cultures can survive subsequent grafting to the adult rat striatum. To further explore the functional efficacy of such grafts, embryonic day 13 ventral mesencephalic tissue was grafted either after 7...

Contested embryonic culture in Japan--public discussion, and human embryonic stem cell research in an aging welfare society.

Science.gov (United States)

Sleeboom-Faulkner, Margaret

2010-01-01

This article explores the reasons for the lack of a broad discussion on bioethical regulation of human embryonic stem cell research (hESR) in Japan and asks why scientists experience difficulties accessing resources for hESR despite the acclaimed indifference of dominant Japanese culture to embryo research. The article shows how various social actors express their views on the embryo and oocyte donation in terms of dominant Japanese culture, foiled against what is regarded as Western culture. Second, it shows how the lack of concern with hESR should be understood in the context of public health policies and communications and bioethics decision making in Japan. Finally, it interprets the meaning of the embryo in the context of Japan as an aging modern welfare society, explaining how policymakers have come to emphasize the urgency of infertility problems over issues around abortion and embryonic life.

Failure of zinc to prevent dysmorphogenesis of cultured rat conceptuses by anti-yolk sac antiserum

International Nuclear Information System (INIS)

Marlow, R.; Freeman, S.J.

1989-01-01

Day 10 rat conceptuses were cultured for 48h in the presence of either cadmium or anti-vesceral yolk sac antiserum (AVYS). Cadmium was embryotoxic at concentrations exceeding 0.25 ug/ml while AVYS caused embryonic dysmorphogenesis, particularly affecting the optic vesicles, at concentrations of 2 ul/ml and above. The effect of pretreatment with zinc on embryotoxicity caused by cadmium or AVYS was studied. Zinc ameliorated the effects of cadmium but had no effect on AVYS-induced embryonic abnormalities. In a second set of experiments inhibition of 125 I-labelled PVP uptake by the yolk sac of cultured whole conceptuses was studied. Cadmium and AVYS both inhibited uptake compared to control cultures. Zinc again ameliorated the effect of cadmium but had no action against AVYS-induced inhibition. These results are in contrast to their previous findings using isolated cultured yolk sacs in which zinc ameliorated the inhibitory effects on 125 I-labelled PVP uptake of both cadmium and AVYS. These data show that in experiments using the isolated cultured yolk sac and the intact cultured conceptus, a qualitatively different response in yolk sac behavior is observed under similar experimental conditions

Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth

Energy Technology Data Exchange (ETDEWEB)

Harrill, Joshua A.; Freudenrich, Theresa M.; Robinette, Brian L.; Mundy, William R., E-mail: mundy.william@epa.gov

2011-11-15

There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth. There was a large increase in neurite outgrowth between 2 and 24 h in both rat and human cultures. Image analysis data collected across multiple cultures demonstrated that neurite outgrowth measurements in rat cortical cultures were more reproducible and had higher dynamic range as compared to human neural cultures. Human neural cultures were more sensitive than rat cortical cultures to chemicals previously shown to inhibit neurite outgrowth. Parallel analysis of morphological (neurite count, neurite length) and cytotoxicity (neurons per field) measurements were used to detect selective effects on neurite outgrowth. All chemicals which inhibited neurite outgrowth in rat cortical cultures did so at concentrations which did not concurrently affect the number of neurons per field, indicating selective effects on neurite outgrowth. In contrast, more than half the chemicals which inhibited neurite outgrowth in human neural cultures did so at concentrations which concurrently decreased the number of neurons per field, indicating that effects on neurite outgrowth were secondary to cytotoxicity. Overall, these data demonstrate that the culture models performed differently in terms of reproducibility, dynamic range and sensitivity to neurite outgrowth inhibitors. While human neural

Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth

International Nuclear Information System (INIS)

Harrill, Joshua A.; Freudenrich, Theresa M.; Robinette, Brian L.; Mundy, William R.

2011-01-01

There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth. There was a large increase in neurite outgrowth between 2 and 24 h in both rat and human cultures. Image analysis data collected across multiple cultures demonstrated that neurite outgrowth measurements in rat cortical cultures were more reproducible and had higher dynamic range as compared to human neural cultures. Human neural cultures were more sensitive than rat cortical cultures to chemicals previously shown to inhibit neurite outgrowth. Parallel analysis of morphological (neurite count, neurite length) and cytotoxicity (neurons per field) measurements were used to detect selective effects on neurite outgrowth. All chemicals which inhibited neurite outgrowth in rat cortical cultures did so at concentrations which did not concurrently affect the number of neurons per field, indicating selective effects on neurite outgrowth. In contrast, more than half the chemicals which inhibited neurite outgrowth in human neural cultures did so at concentrations which concurrently decreased the number of neurons per field, indicating that effects on neurite outgrowth were secondary to cytotoxicity. Overall, these data demonstrate that the culture models performed differently in terms of reproducibility, dynamic range and sensitivity to neurite outgrowth inhibitors. While human neural

Comprehensive Method for Culturing Embryonic Dorsal Root Ganglion Neurons for Seahorse Extracellular Flux XF24 Analysis.

Science.gov (United States)

Lange, Miranda; Zeng, Yan; Knight, Andrew; Windebank, Anthony; Trushina, Eugenia

2012-01-01

Changes in mitochondrial dynamics and function contribute to progression of multiple neurodegenerative diseases including peripheral neuropathies. The Seahorse Extracellular Flux XF24 analyzer provides a comprehensive assessment of the relative state of glycolytic and aerobic metabolism in live cells making this method instrumental in assessing mitochondrial function. One of the most important steps in the analysis of mitochondrial respiration using the Seahorse XF24 analyzer is plating a uniform monolayer of firmly attached cells. However, culturing of primary dorsal root ganglion (DRG) neurons is associated with multiple challenges, including their propensity to form clumps and detach from the culture plate. This could significantly interfere with proper analysis and interpretation of data. We have tested multiple cell culture parameters including coating substrates, culture medium, XF24 microplate plastics, and plating techniques in order to optimize plating conditions. Here we describe a highly reproducible method to obtain neuron-enriched monolayers of securely attached dissociated primary embryonic (E15) rat DRG neurons suitable for analysis with the Seahorse XF24 platform.

Comprehensive method for culturing embryonic dorsal root ganglion neurons for Seahorse Extracellular Flux XF24 Analysis

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Miranda L. Lange

2012-12-01

Full Text Available Changes in mitochondrial dynamics and function contribute to progression of multiple neurodegenerative diseases including peripheral neuropathies. The Seahorse Extracellular Flux XF24 analyzer provides a comprehensive assessment of the relative state of glycolytic and aerobic metabolism in live cells making this method instrumental in assessing mitochondrial function. One of the most important steps in the analysis of mitochondrial respiration using the Seahorse XF24 analyzer is plating a uniform monolayer of firmly attached cells. However, culturing of primary dorsal root ganglion (DRG neurons is associated with multiple challenges, including their propensity to form clumps and detach from the culture plate. This could significantly interfere with proper analysis and interpretation of data. We have tested multiple cell culture parameters including coating substrates, culture medium, XF24 microplate plastics, and plating techniques in order to optimize plating conditions. Here we describe a highly reproducible method to obtain neuron-enriched monolayers of securely attached dissociated primary embryonic (E15 rat DRG neurons suitable for analysis with the Seahorse XF24 platform.

Embryonic Cell Grafts in a Culture Model of Spinal Cord Lesion: Neuronal Relay Formation is Essential for Functional Regeneration

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Anne Tscherter

2016-09-01

Full Text Available Presently there exists no cure for spinal cord injury. However, transplantation of embryonic tissue into spinal cord lesions resulted in axon outgrowth across the lesion site and some functional recovery, fostering hope for future stem cell therapies. Although in vivo evidence for functional recovery is given, the exact cellular mechanism of the graft support remains elusive: either the grafted cells provide a permissive environment for the host tissue to regenerate itself or the grafts actually integrate functionally into the host neuronal network reconnecting the separated spinal cord circuits. We tested the two hypotheses in an in vitro spinal cord lesion model that is based on propagation of activity between two rat organotypic spinal cord slices in culture. Transplantation of dissociated cells from E14 rat spinal cord or forebrain re-established the relay of activity over the lesion site and, thus, provoked functional regeneration. Combining patch-clamp recordings from transplanted cells with network activity measurements from the host tissue on multi-electrode arrays we here show that neurons differentiate from the grafted cells and integrate into the host circuits. Optogenetic silencing of neurons developed from transplanted embryonic mouse forebrain cells provides clear evidence that they replace the lost neuronal connections to relay and synchronize activity between the separated spinal cord circuits. In contrast, transplantation of neurospheres induced neither the differentiation of mature neurons from the grafts nor an improvement of functional regeneration. Together these findings suggest, that the formation of neuronal relays from grafted embryonic cells is essential to re-connect segregated spinal cord circuits.

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro

OpenAIRE

Cong, Shan; Cao, Guifang; Liu, Dongjun

2014-01-01

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic ...

Insulin stimulates choline acetyltransferase activity in cultured embryonic chicken retina neurons

International Nuclear Information System (INIS)

Kyriakis, J.M.; Hausman, R.E.; Peterson, S.W.

1987-01-01

The effect of insulin on the appearance of the enzyme choline acetyltransferase in embryonic chicken retina neurons cultured in defined medium was studied. In the presence of a minimal level of insulin (1 ng/ml), ChoAcT activity increased with time in culture. A correspondence between the insulin concentration in the defined medium (1-100 ng/ml) and both the rate of increase and maximum attained level of ChoAcT activity was observed. Maximal ChoAcT activity was 2- to 3-fold greater in cells cultured in the presence of 100 ng of insulin per ml than in cells cultured in the presence of 1 ng of insulin per ml. To elicit maximum ChoAcT activity, insulin at 100 ng/ml was required in the medium for only the first 4 days of the culture period, at which time insulin could be reduced to maintenance levels (10 ng/ml) without affecting ChoAcT activity. Insulin binding assays performed during a 7-day culture period revealed that irrespective of the 125 I-insulin concentration in the medium during culture, cell-surface insulin receptors decreased by ≅ 90% between 4 and 7 days in culture. This decrease in insulin binding corresponded to the observed decrease in the sensitivity of ChoAcT activity to insulin. The findings suggest that insulin plays a role in mediating cholinergic differentiation in the embryonic chicken retina

Three-dimensional neuroepithelial culture from human embryonic stem cells and its use for quantitative conversion to retinal pigment epithelium.

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Yu Zhu

Full Text Available A goal in human embryonic stem cell (hESC research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.

Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.

Science.gov (United States)

Eiraku, Mototsugu; Sasai, Yoshiki

2011-12-15

Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

Active and passive avoidance conditioning for rats which received x-ray irradiation in their embryonal period

International Nuclear Information System (INIS)

Tamaki, Yoshitaka; Inoue, Minoru; Kameyama, Yoshiro

1983-01-01

Fischer rats at 17 gestational days were given 200 R of x-ray, and their offsprings were subjected to conditioning of active and passive avoidance against a shuttle box stimulation. These rats irradiated in their embryonal period learned active avoidance reaction more rapidly than control rats, but it took time for them to gain passive avoidance reaction. This result seemed to suggest activated reactibility of the irradiated animals in avoiding the shuttle box stimulation. In the irradiated rats, frequency of the passive avoidance reaction increased gradually as they learned with training. (Ueda, J.)

AN EMBRYONIC CHICK PANCREAS ORGAN CULTURE MODEL: CHARACTERIZATION AND NEURAL CONTROL OF EXOCRINE RELEASE

Science.gov (United States)

An embryonic chick (Gallus domesticus) whole-organ pancreas culture system was developed for use as an in vitro model to study cholinergic regulation of exocrine pancreatic function. The culture system was examined for characteristic exocrine function and viability by measuring e...

Adrenal hormones interact with sympathetic innervation to modulate growth of embryonic heart in oculo.

Science.gov (United States)

Tucker, D C; Torres, A

1992-02-01

To allow experimental manipulation of adrenal hormone and autonomic influences on developing myocardium without alteration of hemodynamic load, embryonic rat heart was cultured in the anterior eye chamber of an adult rat. Sympathetic innervation of embryonic day 12 heart grafts was manipulated by surgical sympathectomy of one eye chamber in each host rat. Adrenal hormone exposure was manipulated by host adrenal medullectomy (MEDX) in experiment 1 and by host adrenalectomy (ADX) in experiment 2. In experiment 1, whole heart grafts were larger in MEDX than in sham-operated hosts by 8 wk in oculo (6.14 +/- 0.71 vs. 5.09 +/- 0.69 mm2 with innervation intact and 7.97 +/- 2.07 vs. 3.09 +/- 0.63 mm2 with sympathetic innervation prevented). In experiment 2, host ADX increased growth of embryonic day 12 ventricles grafted into sympathectomized eye chambers (0.69 +/- 0.10 vs. 0.44 +/- 0.04 mm2) but did not affect growth of grafts in intact eye chambers (0.85 +/- 0.09 vs. 1.05 +/- 0.15 mm2). Corticosterone replacement (4 mg/day) entirely reversed the effect of host ADX on graft growth (superior cervical ganglionectomy, 0.47 +/- 0.03 mm2; intact eye chambers, 0.90 +/- 0.91 mm2). Beating rate of grafts was not affected by adrenal hormone manipulations. These experiments indicate that the compromised growth of embryonic heart grafts placed in sympathectomized eye chambers requires exposure to adult levels of glucocorticoids during the early days after grafting. These results suggest that interactions between neural and hormonal stimulation influence cardiac growth in the in oculo culture system and during normal development.

Differential radiosensitivity of mouse embryonic neurons and glia in cell culture

International Nuclear Information System (INIS)

Dambergs, R.; Kidson, C.

1977-01-01

The responses of neurons and glial cells to ultraviolet and γ-radiation were studied in cell cultures of embryonic mouse brains. A decrease in the ratio of glia to neurons occurred after both forms of irradiation. [ 3 H]thymidine labelling followed by autoradiography revealed that all glia were capable of replication, whereas 70 percent of neurons were non-replicating under the conditions of the study. Ultraviolet radiation caused a decrease in the proportion of replicating neurons but did not affect the proportion of replicating glia, whereas γ-radiation caused a decrease in DNA replication in both cell types. Levels of ultraviolet radiation-induced unscheduled DNA synthesis were lower in neurons than in glia. It is concluded that sensitivity to both ionizing and ultraviolet radiation of neurons and glial cells in embryonic brain cultures is determined primarily by the capacity for and state of DNA replication. Neurons which have already reached the stage of terminal differentiation are more resistant than replicating neurons of glial cells

Suppression of decidual cell response induced by tributyltin chloride in pseudopregnant rats: a cause of early embryonic loss

Energy Technology Data Exchange (ETDEWEB)

Harazono, A.; Ema, M. [National Inst. of Health Sciences, Osaka Branch (Japan)

2000-12-01

In our previous studies, tributyltin chloride (TBTC1) at doses of 16.3 mg/kg and above caused implantation failure (preimplantation embryonic loss) and postimplantation embryonic loss in rats following administration on gestational day (GD) 0 through GD 3 and GD 4 through GD 7, respectively. This study was designed to assess the effects of TBTC1 on uterine function as a cause of early embryonic loss in pseudopregnant rats. TBTC1 was given orally to pseudopregnant rats at doses of 4.1, 8.1, 16.3 and 32.5 mg/kg on pseudopregnant day (PPD) 0 to PPD 3 or 8.1, 16.3, 32.5 and 65.1 mg/kg on PPD 4 to PPD 7. The decidual cell response was induced by bilateral scratch trauma on PPD 4. The uterine weight on PPD 9 served as an index of uterine decidualization. Uterine weight and serum progesterone levels on PPD 9 were significantly decreased after administration of TBTC1 at doses of 16.3 mg/kg and above on PPD 0 to PPD 3 or PPD 4 to PPD 7. Administration of TBTC1 at doses of 8.1 mg/kg and above on PPD 0 to 3 also significantly decreased serum progesterone levels on PPD 4. TBTC1 had no effect on ovarian weight and number of corpora lutea. It can be concluded that TBTC1 suppresses the uterine decidual cell response and decreases progesterone levels, and these effects are responsible for early embryonic loss due to TBTC1 exposure. (orig.)

Dynamic 3D culture promotes spontaneous embryonic stem cell differentiation in vitro.

Science.gov (United States)

Gerlach, Jörg C; Hout, Mariah; Edsbagge, Josefina; Björquist, Petter; Lübberstedt, Marc; Miki, Toshio; Stachelscheid, Harald; Schmelzer, Eva; Schatten, Gerald; Zeilinger, Katrin

2010-02-01

Spontaneous in vitro differentiation of mouse embryonic stem cells (mESC) is promoted by a dynamic, three-dimensional (3D), tissue-density perfusion technique with continuous medium perfusion and exchange in a novel four-compartment, interwoven capillary bioreactor. We compared ectodermal, endodermal, and mesodermal immunoreactive tissue structures formed by mESC at culture day 10 with mouse fetal tissue development at gestational day E9.5. The results show that the bioreactor cultures more closely resemble mouse fetal tissue development at gestational day E9.5 than control mESC cultured in Petri dishes.

Involvement of GSK3 in the formation of the leading process and migration of neurons from the embryonic rat medial ganglionic eminence in vitro.

Science.gov (United States)

Niimura, Yuri; Aminaka, Yuichi; Hayashi, Kensuke

2015-03-04

Migrating neurons have leading processes that direct cell movement in response to guidance cues. We investigated the involvement of glycogen synthase kinase 3 (GSK3) in the formation of leading processes and migration of neurons in vitro. We used embryonic rat medial ganglionic eminence (MGE) neurons, which are precursors of inhibitory neurons that migrate into the cerebral cortex. When MGE neurons were placed on an astrocyte layer, they migrated freely with the highest speed among neurons from other parts of the embryonic forebrain. When they were cultured alone, they showed bipolar morphology and extended leading processes within 20 h. Their leading processes had large growth cones, but did not elongate during 3 days in culture, indicating that leading processes are distinct from short axons. Next, we examined the effect of GSK3 inhibitors on leading processes and the migratory behavior of MGE neurons. MGE neurons treated with GSK3 inhibitors showed multipolar morphology and altered process shapes. Moreover, migration of MGE neurons on the astrocyte layer was significantly decreased in the presence of GSK3 inhibitors. These data suggest that GSK3 is involved in the formation of leading processes and in the migration of MGE neurons.

Optimization of a serum-free culture medium for mouse embryonic stem cells using design of experiments (DoE) methodology.

Science.gov (United States)

Knöspel, Fanny; Schindler, Rudolf K; Lübberstedt, Marc; Petzolt, Stephanie; Gerlach, Jörg C; Zeilinger, Katrin

2010-12-01

The in vitro culture behaviour of embryonic stem cells (ESC) is strongly influenced by the culture conditions. Current culture media for expansion of ESC contain some undefined substances. Considering potential clinical translation work with such cells, the use of defined media is desirable. We have used Design of Experiments (DoE) methods to investigate the composition of a serum-free chemically defined culture medium for expansion of mouse embryonic stem cells (mESC). Factor screening analysis according to Plackett-Burman revealed that insulin and leukaemia inhibitory factor (LIF) had a significant positive influence on the proliferation activity of the cells, while zinc and L: -cysteine reduced the cell growth. Further analysis using minimum run resolution IV (MinRes IV) design indicates that following factor adjustment LIF becomes the main factor for the survival and proliferation of mESC. In conclusion, DoE screening assays are applicable to develop and to refine culture media for stem cells and could also be employed to optimize culture media for human embryonic stem cells (hESC).

α-lipoic acid inhibits oxidative stress in testis and attenuates testicular toxicity in rats exposed to carbimazole during embryonic period

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P. Prathima

Full Text Available The aim of this study was to evaluate the probable protective effect of α-lipoic acid against testicular toxicity in rats exposed to carbimazole during the embryonic period. Time-mated pregnant rats were exposed to carbimazole from the embryonic days 9–21. After completion of the gestation period, all the rats were allowed to deliver pups and weaned. At postnatal day 100, F1 male pups were assessed for the selected reproductive endpoints. Gestational exposure to carbimazole decreased the reproductive organ indices, testicular daily sperm count, epididymal sperm variables viz., sperm count, viable sperm, motile sperm and HOS-tail coiled sperms. Significant decrease in the activity levels of 3β- and 17β-hydroxysteroid dehydrogenases and expression of StAR mRNA levels with a significant increase in the total cholesterol levels were observed in the testis of experimental rats over the controls. These events were also accompanied by a significant reduction in the serum testosterone levels in CBZ exposed rats, indicating reduced steroidogenesis. In addition, the deterioration of the testicular architecture and reduced fertility ability were noticed in the carbimazole exposed rats. Significant reduction in the activity levels of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and reduced glutathione content with a significant increase in the levels of lipid peroxidation were observed in the testis of carbimazole exposed rats over the controls. Conversely, supplementation of α-lipoic acid (70 mg/Kg bodyweight ameliorated the male reproductive health in rats exposed to carbimazole during the embryonic period as evidenced by enhanced reproductive organ weights, selected sperm variables, testicular steroidogenesis, and testicular enzymatic and non-enzymatic antioxidants. To conclude, diminished testicular antioxidant balance associated with reduced spermatogenesis and steroidogenesis might be responsible

Transplantation of human neonatal foreskin stromal cells in ex vivo organotypic cultures of embryonic chick femurs

DEFF Research Database (Denmark)

Aldahmash, Abdullah; Vishnubalaji, Radhakrishnan

2017-01-01

NSSCs in ex vivo organotypic cultures of embryonic chick femurs. Isolated embryonic chick femurs (E10 and E11) were cultured for 10 days together with micro-mass cell pellets of hNSSCs, human umbilical vein endothelial cells (HUVEC) or a combination of the two cell types. Changes in femurs gross morphology......We have previously reported that human neonatal foreskin stromal cells (hNSSCs) promote angiogenesis in vitro and in chick embryo chorioallantoic membrane (CAM) assay in vivo. To examine the in vivo relevance of this observation, we examined in the present study the differentiation potential of h......NSSC + HUVEC cultures. Our data suggest that organotypic cultures can be employed to test the differentiation potential of stem cells and demonstrate the importance of stem cell interaction with 3D-intact tissue microenvironment for their differentiation....

Influence of demineralized bone matrix's embryonic origin on bone formation: an experimental study in rats.

Science.gov (United States)

Stavropoulos, Andreas; Kostopoulos, Lambros; Mardas, Nicolaos; Karring, Thorkild

2003-01-01

There are results suggesting that differences regarding bone-inducing potential, in terms of amount and/or rate of bone formation, exist between demineralized bone matrices (DBMs) of different embryonic origins. The aim of the present study was to examine whether the embryonic origin of DBM affects bone formation when used as an adjunct to guided tissue regeneration (GTR). Endomembranous (EM) and endochondral (ECH) DBMs were produced from calvarial and long bones of rats, respectively. Prior to the study the osteoinductive properties of the DBMs were confirmed in six rats following intramuscular implantation. Following surgical exposure of the mandibular ramus, a rigid hemispheric Teflon capsule loosely packed with a standardized quantity of DBM was placed with its open part facing the lateral surface of the ramus in both sides of the jaw in 30 rats. In one side of the jaw, chosen at random, the capsule was filled with EM-DBM, whereas in the other side ECH-DBM was used. Groups of 10 animals were sacrificed after healing periods of 1, 2, and 4 months, and undecalcified sections of the capsules were produced and subjected to histologic analysis and computer-assisted planimetric measurements. During the experiment increasing amounts of newly formed bone were observed inside the capsules in both sides of the animals' jaws. Limited bone formation was observed in the 1- and 2-month specimens, but after 4 months of healing, the newly formed bone in the ECH-DBM grafted sides occupied 59.1% (range 45.6-74.7%) of the area created by the capsule versus 46.9% (range 23.0-64.0%) in the EM-DBM grafted sides (p =.01). It is concluded that the embryonic origin of DBM influences bone formation by GTR and that ECH-DBM is superior to EM-DBM.

Embryonic Stem Cell Culture Conditions Support Distinct States Associated with Different Developmental Stages and Potency

DEFF Research Database (Denmark)

Martin Gonzalez, Javier; Morgani, Sophie M; Bone, Robert A

2016-01-01

. Conversely, the transcriptome of serum-cultured ESCs correlated with later stages of development (E4.5), at which point embryonic cells are more restricted in their developmental potential. Thus, ESC culture systems are not equivalent, but support cell types that resemble distinct developmental stages. Cells...... derived in one condition can be reprogrammed to another developmental state merely by adaptation to another culture condition....

2,3,7,8-Tetrachlorodibenzo-p-dioxin specifically reduces mRNA for the mineralization-related dentin sialophosphoprotein in cultured mouse embryonic molar teeth

International Nuclear Information System (INIS)

Kiukkonen, Anu; Sahlberg, Carin; Lukinmaa, Pirjo-Liisa; Alaluusua, Satu; Peltonen, Eija; Partanen, Anna-Maija

2006-01-01

Previous studies show that the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), interferes with mineralization of the dental matrices in developing mouse and rat teeth. Culture of mouse embryonic molar teeth with TCDD leads to the failure of enamel to be deposited and dentin to undergo mineralization. Lactationally exposed rats show defectively matured enamel and retardation of dentin mineralization. To see if the impaired mineralization is associated with changes in the expression of dentin sialophosphoprotein (Dspp), Bono1 and/or matrix metalloproteinase-20 (MMP-20), thought to be involved in mineralization of the dental hard tissues, we cultured mouse (NMRI) E18 mandibular molars for 3, 5 or 7 days and exposed them to 1 μM TCDD after 2 days of culture. As detected by in situ hybridization of tissue sections, localization and intensity of Bono1 and MMP-20 expression showed no definite difference between the control and exposed tooth explants, suggesting that TCDD does not affect their expression. On the contrary, TCDD reduced or prevented the expression of Dspp in secretory odontoblasts and decreased it in presecretory ameloblasts. The results suggest that the retardation of dentin mineralization by TCDD in mouse molar teeth involves specific interference with Dspp expression

Folic Acid supplementation stimulates notch signaling and cell proliferation in embryonic neural stem cells.

Science.gov (United States)

Liu, Huan; Huang, Guo-Wei; Zhang, Xu-Mei; Ren, Da-Lin; X Wilson, John

2010-09-01

The present study investigated the effect of folic acid supplementation on the Notch signaling pathway and cell proliferation in rat embryonic neural stem cells (NSCs). The NSCs were isolated from E14-16 rat brain and grown as neurospheres in serum-free suspension culture. Individual cultures were assigned to one of 3 treatment groups that differed according to the concentration of folic acid in the medium: Control (baseline folic acid concentration of 4 mg/l), low folic acid supplementation (4 mg/l above baseline, Folate-L) and high folic acid supplementation (40 mg/l above baseline, Folate-H). NSCs were identified by their expression of immunoreactive nestin and proliferating cells by incorporation of 5'bromo-2'deoxyuridine. Cell proliferation was also assessed by methyl thiazolyl tetrazolium assay. Notch signaling was analyzed by real-time PCR and western blot analyses of the expression of Notch1 and hairy and enhancer of split 5 (Hes5). Supplementation of NSCs with folic acid increased the mRNA and protein expression levels of Notch1 and Hes5. Folic acid supplementation also stimulated NSC proliferation dose-dependently. Embryonic NSCs respond to folic acid supplementation with increased Notch signaling and cell proliferation. This mechanism may mediate the effects of folic acid supplementation on neurogenesis in the embryonic nervous system.

Reproductive Toxicity of Zishen Yutai Pill in Rats: The Fertility and Early Embryonic Development Study (Segment I

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Li Zhou

2016-01-01

Full Text Available Purpose. This study was aimed to investigate the reproductive toxicity of Zishen Yutai Pill (ZYP on fertility and early embryonic development in rats. Methods. SD rats were randomly divided into 5 groups: vehicle control group (distilled water, i.g., positive control group (80 mg/kg of cyclophosphamide, i.p., and three ZYP-treated groups (3, 6, and 12 g/kg/d, i.e., 12x, 24x, and 48x clinical doses, i.g.. The high dose was set as the maximum gavage dosage. Results. Cyclophosphamide showed diverse hazards, such as decreased weight of male reproductive organs and sperm density (P<0.05. However, there were no obvious effects of ZYP on physical signs, animal behavior, and survival rate, as well as on weight and food intake during the premating and gestation periods. Importantly, there were no significant adverse effects of ZYP on indexes of copulation, fecundity and fertility indexes, weights and coefficients of male reproductive organs, epididymal sperm number and motility, estrous cycle, preimplantation loss rate, and implantation rate. Besides, the numbers of live and resorbed fetuses per litter were not significantly altered. Conclusions. ZYP had no reproductive toxicities on fertility and early embryonic development in rats at 48x equivalent clinical doses.

Embryonic kidney function in a chronic renal failure model in rodents.

Science.gov (United States)

Fujimoto, Eisuke; Yamanaka, Shuichiro; Kurihara, Sho; Tajiri, Susumu; Izuhara, Luna; Katsuoka, Yuichi; Yokote, Shinya; Matsumoto, Kei; Kobayashi, Eiji; Okano, Hirotaka James; Chikaraishi, Tatsuya; Yokoo, Takashi

2017-08-01

Rapid advancements have been made in alternative treatments for renal diseases. Our goal for renal regeneration is to establish a kidney graft derived from human embryonic tissues. In this study, we investigated the effects of host renal failure on the structure and activity of transplanted embryonic kidney and bladder, and found that diuretics effectively induced urine production in the transplanted kidney. Uremic conditions were reproduced using a 5/6 renal infarction rat model. An embryonic kidney plus bladder (embryonic day 15) was isolated from a pregnant Lewis rat and transplanted into the para-aortic area of a 5/6 renal-infarcted Lewis rat. Following growth, the embryonic bladder was successfully anastomosed to the host ureter. We assessed graft function in terms of survival rates and found no differences between normal (n = 5) and renal failure (n = 8) groups (median survival: 70.5 vs 74.5 h; p = 0.331) in terms of survival, indicating that the grafts prolonged rat survival, even under renal failure conditions. Furosemide (n = 9) significantly increased urine volume compared with saline-treated controls (n = 7; p < 0.05), confirming that the grafts were functional. We also demonstrated the possibilities of an in vivo imaging system for determining the viability of transplanted embryonic kidney with bladder. The results of this study demonstrate that transplanted embryonic kidney and bladder can grow and function effectively, even under uremic conditions.

Concentration-dependent gene expression responses to flusilazole in embryonic stem cell differentiation cultures

International Nuclear Information System (INIS)

Dartel, Dorien A.M. van; Pennings, Jeroen L.A.; Fonteyne, Liset J.J. de la; Brauers, Karen J.J.; Claessen, Sandra; Delft, Joost H. van; Kleinjans, Jos C.S.; Piersma, Aldert H.

2011-01-01

The murine embryonic stem cell test (EST) is designed to evaluate developmental toxicity based on compound-induced inhibition of embryonic stem cell (ESC) differentiation into cardiomyocytes. The addition of transcriptomic evaluation within the EST may result in enhanced predictability and improved characterization of the applicability domain, therefore improving usage of the EST for regulatory testing strategies. Transcriptomic analyses assessing factors critical for risk assessment (i.e. dose) are needed to determine the value of transcriptomic evaluation in the EST. Here, using the developmentally toxic compound, flusilazole, we investigated the effect of compound concentration on gene expression regulation and toxicity prediction in ESC differentiation cultures. Cultures were exposed for 24 h to multiple concentrations of flusilazole (0.54-54 μM) and RNA was isolated. In addition, we sampled control cultures 0, 24, and 48 h to evaluate the transcriptomic status of the cultures across differentiation. Transcriptomic profiling identified a higher sensitivity of development-related processes as compared to cell division-related processes in flusilazole-exposed differentiation cultures. Furthermore, the sterol synthesis-related mode of action of flusilazole toxicity was detected. Principal component analysis using gene sets related to normal ESC differentiation was used to describe the dynamics of ESC differentiation, defined as the 'differentiation track'. The concentration-dependent effects on development were reflected in the significance of deviation of flusilazole-exposed cultures from this transcriptomic-based differentiation track. Thus, the detection of developmental toxicity in EST using transcriptomics was shown to be compound concentration-dependent. This study provides further insight into the possible application of transcriptomics in the EST as an improved alternative model system for developmental toxicity testing.

Generation of Functional Thyroid Tissue Using 3D-Based Culture of Embryonic Stem Cells.

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Antonica, Francesco; Kasprzyk, Dominika Figini; Schiavo, Andrea Alex; Romitti, Mírian; Costagliola, Sabine

2017-01-01

During the last decade three-dimensional (3D) cultures of pluripotent stem cells have been intensively used to understand morphogenesis and molecular signaling important for the embryonic development of many tissues. In addition, pluripotent stem cells have been shown to be a valid tool for the in vitro modeling of several congenital or chronic human diseases, opening new possibilities to study their physiopathology without using animal models. Even more interestingly, 3D culture has proved to be a powerful and versatile tool to successfully generate functional tissues ex vivo. Using similar approaches, we here describe a protocol for the generation of functional thyroid tissue using mouse embryonic stem cells and give all the details and references for its characterization and analysis both in vitro and in vivo. This model is a valid approach to study the expression and the function of genes involved in the correct morphogenesis of thyroid gland, to elucidate the mechanisms of production and secretion of thyroid hormones and to test anti-thyroid drugs.

Bioprotective effect of zinc in macro- and nanoaquachelate form on embryonal development of rats in conditions of lead intoxication

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Beletskaya E.M.

2013-06-01

Full Text Available The article presents results of studied influence of low doses of lead and zinc (nanozinc on embryonal development in a la¬boratory experiment on rats. Negative influence of lead on pregnancy of laboratory animals, manifested in violation of the physiological dynamics of the rectal temperature and decrease in body weight gain was revealed. Embryotoxic effect of low doses of lead results in increased fetal mortality by 2.16 times compared to the control group of animals, de¬terioration of the morphometric indices of fetuses, violation of placentogenesis. Simultaneous injections of zinc on back¬ground of lead intoxication causes a protective effect on the body of pregnant rats and embryonal development of the offspring, more pronounced for zinc citrate, received by using aquananotehnology, as compared to zinc chloride. Thus, by morphometry indices, male fetuses were more sensitive to prenatal lead exposure in comparison to female fetuses.

Adult and embryonic GAD transcripts are spatiotemporally regulated during postnatal development in the rat brain.

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Anke Popp

Full Text Available BACKGROUND: GABA (gamma-aminobutyric acid, the main inhibitory neurotransmitter in the brain, is synthesized by glutamic acid decarboxylase (GAD. GAD exists in two adult isoforms, GAD65 and GAD67. During embryonic brain development at least two additional transcripts exist, I-80 and I-86, which are distinguished by insertions of 80 or 86 bp into GAD67 mRNA, respectively. Though it was described that embryonic GAD67 transcripts are not detectable during adulthood there are evidences suggesting re-expression under certain pathological conditions in the adult brain. In the present study we systematically analyzed for the first time the spatiotemporal distribution of different GADs with emphasis on embryonic GAD67 mRNAs in the postnatal brain using highly sensitive methods. METHODOLOGY/PRINCIPAL FINDINGS: QPCR was used to precisely investigate the postnatal expression level of GAD related mRNAs in cortex, hippocampus, cerebellum, and olfactory bulb of rats from P1 throughout adulthood. Within the first three postnatal weeks the expression of both GAD65 and GAD67 mRNAs reached adult levels in hippocampus, cortex, and cerebellum. The olfactory bulb showed by far the highest expression of GAD65 as well as GAD67 transcripts. Embryonic GAD67 splice variants were still detectable at birth. They continuously declined to barely detectable levels during postnatal development in all investigated regions with exception of a comparatively high expression in the olfactory bulb. Radioactive in situ hybridizations confirmed the occurrence of embryonic GAD67 transcripts in the olfactory bulb and furthermore detected their localization mainly in the subventricular zone and the rostral migratory stream. CONCLUSIONS/SIGNIFICANCE: Embryonic GAD67 transcripts can hardly be detected in the adult brain, except for specific regions associated with neurogenesis and high synaptic plasticity. Therefore a functional role in processes like proliferation, migration or

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats

OpenAIRE

Matveyenko, Aleksey V.; Georgia, Senta; Bhushan, Anil; Butler, Peter C.

2010-01-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in ...

Bone matrix calcification during embryonic and postembryonic rat calvarial development assessed by SEM-EDX spectroscopy, XRD, and FTIR spectroscopy.

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Henmi, Akiko; Okata, Hiroshi; Anada, Takahisa; Yoshinari, Mariko; Mikami, Yasuto; Suzuki, Osamu; Sasano, Yasuyuki

2016-01-01

Bone mineral is constituted of biological hydroxyapatite crystals. In developing bone, the mineral crystal matures and the Ca/P ratio increases. However, how an increase in the Ca/P ratio is involved in maturation of the crystal is not known. The relationships among organic components and mineral changes are also unclear. The study was designed to investigate the process of calcification during rat calvarial bone development. Calcification was evaluated by analyzing the atomic distribution and concentration of Ca, P, and C with scanning electron microscopy (SEM)-energy-dispersive X-ray (EDX) spectroscopy and changes in the crystal structure with X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. Histological analysis showed that rat calvarial bone formation started around embryonic day 16. The areas of Ca and P expanded, matching the region of the developing bone matrix, whereas the area of C became localized around bone. X-ray diffraction and FTIR analysis showed that the amorphous-like structure of the minerals at embryonic day 16 gradually transformed into poorly crystalline hydroxyapatite, whereas the proportion of mineral to protein increased until postnatal week 6. FTIR analysis also showed that crystallization of hydroxyapatite started around embryonic day 20, by which time SEM-EDX spectroscopy showed that the Ca/P ratio had increased and the C/Ca and C/P ratios had decreased significantly. The study suggests that the Ca/P molar ratio increases and the proportion of organic components such as proteins of the bone matrix decreases during the early stage of calcification, whereas crystal maturation continues throughout embryonic and postembryonic bone development.

Long-term culture of chicken primordial germ cells isolated from embryonic blood and production of germline chimaeric chickens.

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Naito, Mitsuru; Harumi, Takashi; Kuwana, Takashi

2015-02-01

Production of germline chimaeric chickens by the transfer of cultured primordial germ cells (PGC) is a useful system for germline manipulation. A novel culture system was developed for chicken PGC isolated from embryonic blood. The isolated PGC were cultured on feeder cells derived from chicken embryonic fibroblast. The cultured PGC formed colonies and they proliferated about 300-times during the first 30 days. The cultured PGC retained the ability to migrate to recipient gonads and were also chicken VASA homologue (CVH)-positive. Female PGC were present in the mixed-sex PGC populations cultured for more than 90 days and gave rise to viable offspring efficiently via germline chimaeric chickens. Male cultured PGC were transferred to recipient embryos and produced putative chimaeric chickens. The DNA derived from the cultured PGC was detected in the sperm samples of male putative chimaeric chickens, but no donor derived offspring were obtained. Donor-derived offspring were also obtained from germline chimaeric chickens by the transfer of frozen-thawed cultured PGC. The culture method for PGC developed in the present study is useful for manipulation of the germline in chickens, such as preservation of genetic resources and gene transfer. Copyright © 2014 Elsevier B.V. All rights reserved.

mRNA Fragments in In-Vitro Culture Media are Associated with Bovine Preimplantation Embryonic Development

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Jenna eKropp

2015-08-01

Full Text Available In vitro production (IVP systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerated conditioned media. Differential expression was confirmed by quantitative real-time PCR for

Culturing of primary rat neurons and glia on ultra-thin parylene-C

International Nuclear Information System (INIS)

Unsworth, C.P.; Delivopoulos, E.; Murray, A.F.

2010-01-01

Full text: In this article, we will describe how we have successfully cultured dissociated embryonic cortical neurons and glia from the postnatal rat hippocampus on extremely thin layers (up to 10 nm) of Parylene-C on a silicon dioxide substrate. Silicon wafers were oxidised, deposited with the biomaterial, Parylene-C, photo-lithographically patterned and plasma etched to produce chips that consisted of lines of Paryl ene-C with varying widths, thickness and lengths. The chips produced were then immersed in Horse Serum and plated with the cells. Ratios of Neurons; Glia; Cell Body were measured on, adjacent to and away from the Parylene-C. Our initial results show how these ratios remained roughly constant for ultra-thin Parylene-C thicknesses of 10 nm as compared to a benchmark thickness of 100 nm (where such cells are known to grow well). Thus, our findings demonstrate that it is possible to culture primary rat neurons and glia to practically cell membrane thicknesses of Parylene-C. Being able to culture cells on such ultra thin levels of Parylene-C will open up the possibility to develop Multi-Electrode Arrays (MEA) that can capacitively couple embedded electrodes through the parylene to the cells on its surface. Thus, providing a neat, insulated passive electrode. Only the ultra-thin thicknesses of Parylene demonstrated here would allow for the rea isation of such a technology. Hence, the outcome of this work, will be of great interest to the Neuroengineering and the Multi-Electrode Array (MEA) community, as an alternative material for the fabric tion of passive electrodes, used in capacitive coupling mode.

Beta-hydroxybutyrate increases reactive oxygen species in late but not in early postimplantation embryonic cells in vitro.

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Forsberg, H; Eriksson, U J; Melefors, O; Welsh, N

1998-02-01

Embryonic dysmorphogenesis has been blocked by antioxidant treatment in vivo and in vitro, suggesting that embryonic excess of reactive oxygen species (ROS) has a role in the teratogenic process of diabetic pregnancy. We report that the basal levels of ROS in dispersed rat embryonic cells in vitro, as determined by fluorescence of dichlorofluorescein (DCF), were not different in cells from control and diabetic pregnancy at day 10 or 12. Beta-hydroxybutyrate (beta-HB) and succinic acid monomethyl ester both augmented DCF fluorescence in cells from day 12 embryos of normal and diabetic rats but not from day 10 embryos. Cells of day 10 and day 12 embryos from normal and diabetic rats responded to increasing glucose concentrations with a dosage-dependent alleviation of DCF fluorescence. Day 10 embryonic cells exhibited high glucose utilization rates and high pentose phosphate shunt rates, but low mitochondrial oxidation rates. Moreover, in vitro culture of embryos between gestational days 9 and 10 in the presence of 20% oxygen induced an increased and glucose-sensitive oxidation of glucose compared with embryos not cultured in vitro. At gestation day 12, however, pentose phosphate shunt rates showed a decrease, whereas the mitochondrial beta-HB oxidation rates were increased compared with those at gestation day 10. This was paralleled by a lower expression of glucose 6-phosphate dehydrogenase- and phosphofructokinase-mRNA levels at day 12 than at day 10. On the other hand, H-ferritin mRNA expression at day 12 was high compared with day 10. None of the mRNA species investigated were affected by the diabetic state of the mother. It was concluded that beta-HB-induced stimulation of mitochondrial oxidative events may lead to the generation of ROS at gestational day 12, but probably not at day 10, when only a minute amount of mitochondrial activity occurs. Thus our results do not support the notion of diabetes-induced mitochondrial oxidative stress before the development of

Embryonic stem cells in scaffold-free three-dimensional cell culture: osteogenic differentiation and bone generation.

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Handschel, Jörg; Naujoks, Christian; Depprich, Rita; Lammers, Lydia; Kübler, Norbert; Meyer, Ulrich; Wiesmann, Hans-Peter

2011-07-14

Extracorporeal formation of mineralized bone-like tissue is still an unsolved challenge in tissue engineering. Embryonic stem cells may open up new therapeutic options for the future and should be an interesting model for the analysis of fetal organogenesis. Here we describe a technique for culturing embryonic stem cells (ESCs) in the absence of artificial scaffolds which generated mineralized miromasses. Embryonic stem cells were harvested and osteogenic differentiation was stimulated by the addition of dexamethasone, ascorbic acid, and ß-glycerolphosphate (DAG). After three days of cultivation microspheres were formed. These spherical three-dimensional cell units showed a peripheral zone consisting of densely packed cell layers surrounded by minerals that were embedded in the extracellular matrix. Alizarine red staining confirmed evidence of mineralization after 10 days of DAG stimulation in the stimulated but not in the control group. Transmission electron microscopy demonstrated scorching crystallites and collagenous fibrils as early indication of bone formation. These extracellular structures resembled hydroxyl apatite-like crystals as demonstrated by distinct diffraction patterns using electron diffraction analysis. The micromass culture technique is an appropriate model to form three-dimensional bone-like micro-units without the need for an underlying scaffold. Further studies will have to show whether the technique is applicable also to pluripotent stem cells of different origin. © 2011 Handschel et al; licensee BioMed Central Ltd.

Embryonic stem cells in scaffold-free three-dimensional cell culture: osteogenic differentiation and bone generation

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Meyer Ulrich

2011-07-01

Full Text Available Abstract Extracorporeal formation of mineralized bone-like tissue is still an unsolved challenge in tissue engineering. Embryonic stem cells may open up new therapeutic options for the future and should be an interesting model for the analysis of fetal organogenesis. Here we describe a technique for culturing embryonic stem cells (ESCs in the absence of artificial scaffolds which generated mineralized miromasses. Embryonic stem cells were harvested and osteogenic differentiation was stimulated by the addition of dexamethasone, ascorbic acid, and ß-glycerolphosphate (DAG. After three days of cultivation microspheres were formed. These spherical three-dimensional cell units showed a peripheral zone consisting of densely packed cell layers surrounded by minerals that were embedded in the extracellular matrix. Alizarine red staining confirmed evidence of mineralization after 10 days of DAG stimulation in the stimulated but not in the control group. Transmission electron microscopy demonstrated scorching crystallites and collagenous fibrils as early indication of bone formation. These extracellular structures resembled hydroxyl apatite-like crystals as demonstrated by distinct diffraction patterns using electron diffraction analysis. The micromass culture technique is an appropriate model to form three-dimensional bone-like micro-units without the need for an underlying scaffold. Further studies will have to show whether the technique is applicable also to pluripotent stem cells of different origin.

Culture in embryonic kidney serum and xeno-free media as renal cell carcinoma and renal cell carcinoma cancer stem cells research model.

Science.gov (United States)

Krawczyk, Krzysztof M; Matak, Damian; Szymanski, Lukasz; Szczylik, Cezary; Porta, Camillo; Czarnecka, Anna M

2018-04-01

The use of fetal bovine serum hinders obtaining reproducible experimental results and should also be removed in hormone and growth factor studies. In particular hormones found in FBS act globally on cancer cell physiology and influence transcriptome and metabolome. The aim of our study was to develop a renal carcinoma serum free culture model optimized for (embryonal) renal cells in order to select the best study model for downstream auto-, para- or endocrine research. Secondary aim was to verify renal carcinoma stem cell culture for this application. In the study, we have cultured renal cell carcinoma primary tumour cell line (786-0) as well as human kidney cancer stem cells in standard 2D monolayer cultures in Roswell Park Memorial Institute Medium or Dulbecco's Modified Eagle's Medium and Complete Human Kidney Cancer Stem Cell Medium, respectively. Serum-free, animal-component free Human Embryonic Kidney 293 media were tested. Our results revealed that xeno-free embryonal renal cells optimized culture media provide a useful tool in RCC cancer biology research and at the same time enable effective growth of RCC. We propose bio-mimic RCC cell culture model with specific serum-free and xeno-free medium that promote RCC cell viability.

Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions

DEFF Research Database (Denmark)

Morgani, Sophie M; Canham, Maurice A; Nichols, Jennifer

2013-01-01

Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought...... not directly support Nanog-positive epiblast-like ESCs. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants....

A voltage-sensitive dye-based assay for the identification of differentiated neurons derived from embryonic neural stem cell cultures.

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Richardson N Leão

Full Text Available BACKGROUND: Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC and embryonic neural stem cell (NSC cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca(2+-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate. METHODOLOGY/PRINCIPAL FINDINGS: In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD. We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters. CONCLUSIONS/SIGNIFICANCE: Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture.

Locust bean gum as an alternative polymeric coating for embryonic stem cell culture

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Perestrelo, Ana Rubina [Regenerative Medicine Program, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve (Portugal); IBB - Institute for Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine (CBME), Universidade do Algarve (Portugal); PhD Program in Biomedical Sciences, Universidade do Algarve (Portugal); Grenha, Ana [IBB - Institute for Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine (CBME), Universidade do Algarve (Portugal); Rosa da Costa, Ana M. [Centro de Investigação em Química do Algarve (CIQA) and Departamento de Química e Farmácia, Faculdade de Ciências e Tecnologia, Universidade do Algarve (Portugal); Belo, José António, E-mail: jose.belo@fcm.unl.pt [Regenerative Medicine Program, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve (Portugal); IBB - Institute for Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine (CBME), Universidade do Algarve (Portugal); Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Campo Mártires da Pátria 130, 1169-056 Lisboa (Portugal)

2014-07-01

Pluripotent embryonic stem cells (ESCs) have self-renewal capacity and the potential to differentiate into any cellular type depending on specific cues (pluripotency) and, therefore, have become a vibrant research area in the biomedical field. ESCs are usually cultured in gelatin or on top of a monolayer of feeder cells such as mitotically inactivated mouse embryonic fibroblasts (MEFsi). The latter is the gold standard support to maintain the ESCs in the pluripotent state. Examples of versatile, non-animal derived and inexpensive materials that are able to support pluripotent ESCs are limited. Therefore, our aim was to find a biomaterial able to support ESC growth in a pluripotent state avoiding laborious and time consuming parallel culture of MEFsi and as simple to handle as gelatin. Many of the new biomaterials used to develop stem cell microenvironments are using natural polymers adsorbed or covalently attached to the surface to improve the biocompatibility of synthetic polymers. Locust beam gum (LBG) is a natural, edible polymer, which has a wide range of potential applications in different fields, such as food and pharmaceutical industry, due to its biocompatibility, adhesiveness and thickening properties. The present work brings a natural system based on the use of LBG as a coating for ESC culture. Undifferentiated mouse ESCs were cultured on commercially available LBG to evaluate its potential in maintaining pluripotent ESCs. In terms of morphology, ESC colonies in LBG presented the regular dome shape with bright borders, similar to the colonies obtained in co-cultures with MEFsi and characteristic of pluripotent ESC colonies. In short-term cultures, ESC proliferation in LBG coating was similar to ESC cultured in gelatin and the cells maintained their viability. The activity of alkaline phosphatase and Nanog, Sox2 and Oct4 expression of mouse ESCs cultured in LBG were comparable or in some cases higher than in ESCs cultured in gelatin. An in vitro

Locust bean gum as an alternative polymeric coating for embryonic stem cell culture

International Nuclear Information System (INIS)

Perestrelo, Ana Rubina; Grenha, Ana; Rosa da Costa, Ana M.; Belo, José António

2014-01-01

Pluripotent embryonic stem cells (ESCs) have self-renewal capacity and the potential to differentiate into any cellular type depending on specific cues (pluripotency) and, therefore, have become a vibrant research area in the biomedical field. ESCs are usually cultured in gelatin or on top of a monolayer of feeder cells such as mitotically inactivated mouse embryonic fibroblasts (MEFsi). The latter is the gold standard support to maintain the ESCs in the pluripotent state. Examples of versatile, non-animal derived and inexpensive materials that are able to support pluripotent ESCs are limited. Therefore, our aim was to find a biomaterial able to support ESC growth in a pluripotent state avoiding laborious and time consuming parallel culture of MEFsi and as simple to handle as gelatin. Many of the new biomaterials used to develop stem cell microenvironments are using natural polymers adsorbed or covalently attached to the surface to improve the biocompatibility of synthetic polymers. Locust beam gum (LBG) is a natural, edible polymer, which has a wide range of potential applications in different fields, such as food and pharmaceutical industry, due to its biocompatibility, adhesiveness and thickening properties. The present work brings a natural system based on the use of LBG as a coating for ESC culture. Undifferentiated mouse ESCs were cultured on commercially available LBG to evaluate its potential in maintaining pluripotent ESCs. In terms of morphology, ESC colonies in LBG presented the regular dome shape with bright borders, similar to the colonies obtained in co-cultures with MEFsi and characteristic of pluripotent ESC colonies. In short-term cultures, ESC proliferation in LBG coating was similar to ESC cultured in gelatin and the cells maintained their viability. The activity of alkaline phosphatase and Nanog, Sox2 and Oct4 expression of mouse ESCs cultured in LBG were comparable or in some cases higher than in ESCs cultured in gelatin. An in vitro

The effects of low dose ionizing radiation on the development of rat cerebral cortex, (2)

International Nuclear Information System (INIS)

Matsushita, Koji

1993-01-01

In order to study the molecular mechanisms of neuronal migration on developing rat cerebral cortex, we need a tissue culture system in which neuronal migration can be observed. We prepared a tissue culture system of embryonic rat cerebral cortex starting on embryonic day 16 and cultivating it for 48 hours. The autoradiographic study in this system revealed not only the migration of 3 H-thymidine labeled neurons but also neuronal migration delays from low doses of ionizing radiation of more than 10 cGy. In addition, on immunohistochemical study, cell-cell adhesion molecule N-CAM staining was remarkably decreased in the matrix cell layer. In the tissue culture system where monoclonal anti-N-CAM antibodies were added, neuronal migration delay comparable to that of 20 cGy radiation was found. In conclusion, it was speculated that neuronal migration delay might be caused by disturbed N-CAM synthesis in matrix cells after low dose ionizing radiation. (author)

Characterization of organotypic ventral mesencephalic cultures from embryonic mice and protection against MPP toxicity by GDNF

DEFF Research Database (Denmark)

Jakobsen, B; Gramsbergen, J B; Møller Dall, A

2005-01-01

We characterized organotypic ventral mesencephalic (VM) cultures derived from embryonic day 12 (E12) mice (CBL57/bL6) in terms of number of dopaminergic neurons, cell soma size and dopamine production in relation to time in vitro and tested the effects of 1-methyl-4-phenylpyridinium (MPP(+)) and ...

Freezing tolerance of sea urchin embryonic cells: Differentiation commitment and cytoskeletal disturbances in culture.

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Odintsova, Nelly A; Ageenko, Natalya V; Kipryushina, Yulia O; Maiorova, Mariia A; Boroda, Andrey V

2015-08-01

This study focuses on the freezing tolerance of sea urchin embryonic cells. To significantly reduce the loss of physiological activity of these cells that occurs after cryopreservation and to study the effects of ultra-low temperatures on sea urchin embryonic cells, we tested the ability of the cells to differentiate into spiculogenic or pigment directions in culture, including an evaluation of the expression of some genes involved in pigment differentiation. A morphological analysis of cytoskeletal disturbances after freezing in a combination of penetrating (dimethyl sulfoxide and ethylene glycol) and non-penetrating (trehalose and polyvinylpyrrolidone) cryoprotectants revealed that the distribution pattern of filamentous actin and tubulin was similar to that in the control cultures. In contrast, very rare spreading cells and a small number of cells with filamentous actin and tubulin were detected after freezing in the presence of only non-penetrating cryoprotectants. The largest number of pigment cells was found in cultures frozen with trehalose or trehalose and dimethyl sulfoxide. The ability to induce the spicule formation was lost in the cells frozen only with non-penetrating cryoprotectants, while it was maximal in cultures frozen in a cryoprotective mixture containing both non-penetrating and penetrating cryoprotectants (particularly, when ethylene glycol was present). Using different markers for cell state assessment, an effective cryopreservation protocol for sea urchin cells was developed: three-step freezing with a low cooling rate (1-2°C/min) and a combination of non-penetrating and penetrating cryoprotectants made it possible to obtain a high level of cell viability (up to 65-80%). Copyright © 2015 Elsevier Inc. All rights reserved.

Comparative Analysis of Human and Rodent Brain Primary Neuronal Culture Spontaneous Activity Using Micro-Electrode Array Technology.

Science.gov (United States)

Napoli, Alessandro; Obeid, Iyad

2016-03-01

Electrical activity in embryonic brain tissue has typically been studied using Micro Electrode Array (MEA) technology to make dozens of simultaneous recordings from dissociated neuronal cultures, brain stem cell progenitors, or brain slices from fetal rodents. Although these rodent neuronal primary culture electrical properties are mostly investigated, it has not been yet established to what extent the electrical characteristics of rodent brain neuronal cultures can be generalized to those of humans. A direct comparison of spontaneous spiking activity between rodent and human primary neurons grown under the same in vitro conditions using MEA technology has never been carried out before and will be described in the present study. Human and rodent dissociated fetal brain neuronal cultures were established in-vitro by culturing on a glass grid of 60 planar microelectrodes neurons under identical conditions. Three different cultures of human neurons were produced from tissue sourced from a single aborted fetus (at 16-18 gestational weeks) and these were compared with seven different cultures of embryonic rat neurons (at 18 gestational days) originally isolated from a single rat. The results show that the human and rodent cultures behaved significantly differently. Whereas the rodent cultures demonstrated robust spontaneous activation and network activity after only 10 days, the human cultures required nearly 40 days to achieve a substantially weaker level of electrical function. These results suggest that rat neuron preparations may yield inferences that do not necessarily transfer to humans. © 2015 Wiley Periodicals, Inc.

Long-term organ culture of adult rat colon

DEFF Research Database (Denmark)

Shamsuddin, A.K.M.; Barrett, L.A.; Autrup, Herman

1978-01-01

. The effect of in vivo carcinogen pretreatment was also studied. The explant culture from control untreated animals showed good epithelial differentiation with crypts until 6 weeks. In contrast, the explants from animals pretreated with 4 weekly doses of azoxymethane consistently showed epithelial......Colon explants from adult rats were maintained in culture for over 3 months in our laboratories with good epithelial preservation and cellular differentiation. The light and transmission electron microscopic features of rat colon mucosa during the culture period are described. In all the explants...

Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture

International Nuclear Information System (INIS)

Miller-Pinsler, Lutfiya; Wells, Peter G.

2015-01-01

Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat b /J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1), exposed for 24 h to 2 or 4 mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p < 0.001). Maternal pretreatment of C57BL/6 WT dams with 50 kU/kg PEG-catalase (PEG-cat) 8 h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p < 0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p < 0.01), and trends for reduced anterior neuropore closure, turning and crown–rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p < 0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies. - Highlights: • Ethanol (EtOH) exposure causes structural embryopathies in embryo culture. • Genetically enhanced catalase (hCat) protects against EtOH embryopathies. • Genetically deficient catalase (aCat) exacerbates EtOH embryopathies. • Embryonic catalase is developmentally important. • EtOH developmental

Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture

Energy Technology Data Exchange (ETDEWEB)

Miller-Pinsler, Lutfiya [Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Wells, Peter G., E-mail: pg.wells@utoronto.ca [Division of Biomolecular Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario (Canada); Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada)

2015-09-15

Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat{sup b}/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1), exposed for 24 h to 2 or 4 mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p < 0.001). Maternal pretreatment of C57BL/6 WT dams with 50 kU/kg PEG-catalase (PEG-cat) 8 h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p < 0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p < 0.01), and trends for reduced anterior neuropore closure, turning and crown–rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p < 0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies. - Highlights: • Ethanol (EtOH) exposure causes structural embryopathies in embryo culture. • Genetically enhanced catalase (hCat) protects against EtOH embryopathies. • Genetically deficient catalase (aCat) exacerbates EtOH embryopathies. • Embryonic catalase is developmentally important. • Et

Isolation and culture of neural crest cells from embryonic murine neural tube.

Science.gov (United States)

Pfaltzgraff, Elise R; Mundell, Nathan A; Labosky, Patricia A

2012-06-02

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types. NC also has the unique ability to influence the differentiation and maturation of target organs. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. NC multipotency was first described from explants of the avian neural tube. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter. The method presented here is adapted from

Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials

Directory of Open Access Journals (Sweden)

Md Amranul Haque, Masato Nagaoka, Bayar Hexig and Toshihiro Akaike

2010-01-01

Full Text Available Nanobiomaterials can play a central role in regenerative medicine and tissue engineering by facilitating cellular behavior and function, such as those where extracellular matrices (ECMs direct embryonic stem (ES cell morphogenesis, proliferation, differentiation and apoptosis. However, controlling ES cell proliferation and differentiation using matrices from natural sources is still challenging due to complex and heterogeneous culture conditions. Moreover, the systemic investigation of the regulation of self-renewal and differentiation to lineage specific cells depends on the use of defined and stress-free culture conditions. Both goals can be achieved by the development of biomaterial design targeting ECM or growth factors for ES cell culture. This targeted application will benefit from expansion of ES cells for transplantation, as well as the production of a specific differentiated cell type either by controlling the differentiation in a very specific pathway or by elimination of undesirable cell types.

Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials

International Nuclear Information System (INIS)

Haque, Md Amranul; Nagaoka, Masato; Hexig, Bayar; Akaike, Toshihiro

2010-01-01

Nanobiomaterials can play a central role in regenerative medicine and tissue engineering by facilitating cellular behavior and function, such as those where extracellular matrices (ECMs) direct embryonic stem (ES) cell morphogenesis, proliferation, differentiation and apoptosis. However, controlling ES cell proliferation and differentiation using matrices from natural sources is still challenging due to complex and heterogeneous culture conditions. Moreover, the systemic investigation of the regulation of self-renewal and differentiation to lineage specific cells depends on the use of defined and stress-free culture conditions. Both goals can be achieved by the development of biomaterial design targeting ECM or growth factors for ES cell culture. This targeted application will benefit from expansion of ES cells for transplantation, as well as the production of a specific differentiated cell type either by controlling the differentiation in a very specific pathway or by elimination of undesirable cell types. (topical review)

Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture.

Science.gov (United States)

Miller-Pinsler, Lutfiya; Wells, Peter G

2015-09-15

Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat(b)/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug=GD 1), exposed for 24h to 2 or 4mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (pcatalase (PEG-cat) 8h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (pcatalase is a determinant of risk for EtOH embryopathies. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of organically bound tritium (OBT) on cultured midbrain cells from embryonic mice

International Nuclear Information System (INIS)

Wang Bing; Akihiro Shima; Takeshi Yamada; Keiko Watganabe

1997-01-01

Objective: Four kinds of organically bound tritium compounds (OBT s ) including 3 H-thymidine, 3 H-uridine, 3 H-arginine and 3 H-glutamic acid, were investigated on proliferation and differentiation of cultured mouse embryonic midbrain cells (MBCs). Methods: MBCs were isolated from day 11 embryos, cultured at a high concentration with the medium containing OBT. Results: Differentiation of MBC was more sensitive to radiation than proliferation. Dose-dependent decrease of DNA and protein contents were also observed. The RBE values, ranging from 4.6 to 8.7, of β rays from OBTs were obtained when compared with X-irradiation at their ID50s (inhibitory dose that reduced assessment value by 50% of the control) on inhibition of cell proliferation and differentiation, and on reduction of DNA and protein contents of the cultures. The mixed exposure to X-rays and one kind of OBTs or to any two kinds of OBTs resulted in more efficiently inhibitory effect on differentiation. Conclusions: MBC culture system was more sensitive to beta radiation from OBTs than to X-rays, which resulted in very high RBE values

mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

Science.gov (United States)

Kropp, Jenna; Khatib, Hasan

2015-01-01

In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

Peculiarities of Embryonic and Post-Embryonic Development of Оesophagostomum dentatum (Nematoda, Strongylidae Larvae Cultured in Vitro

Directory of Open Access Journals (Sweden)

Yevstafieva V. А.

2017-02-01

Full Text Available Morphometric peculiarities of the development of Оesophagostomum dentatum Rudolphi, 1803 from egg to infective larva were studied under laboratory conditions at various temperatures. The determined optimum temperature for embryonic and post-embryonic development of О. dentatum larvae from domestic pig (Sus scrofa domesticus Linnaeus, 1758 is 22 °С. At this temperature, 81 % of larvae develop to the third stage (L3 on the 10th day. Temperatures of 24 °С and 20 °С are less favorable for the development of the nematode, at those temperatures only 67 and 63 % of larvae, respectively, reached infective stage by the 10th day of cultivation. Embryonic development of О. dentatum eggs is characterized by their lengthening (by 8.87-9.50 %, р < 0.01 and widening (by 6.77-9.35 %, р < 0.05-0.01, and post-embryonic larval development is associated with lengthening (by 4.59-17.33 %, р < 0.01-0.001.

Development of striatal patch/matrix organization in organotypic co-cultures of perinatal striatum, cortex and substantia nigra.

Science.gov (United States)

Snyder-Keller, A; Costantini, L C; Graber, D J

2001-01-01

Organotypic cultures of fetal or early postnatal striatum were used to assess striatal patch formation and maintenance in the presence or absence of dopaminergic and glutamatergic influences. Vibratome-cut slices of the striatum prepared from embryonic day 19 to postnatal day 4 rat pups were maintained in static culture on clear membrane inserts in Dulbecco's modified Eagle's medium/F12 (1:1) with 20% horse serum. Some were co-cultured with embryonic day 12-16 ventral mesencephalon and/or embryonic day 19 to postnatal day 4 cortex, which produced a dense dopaminergic innervation and a modest cortical innervation. Donors of striatal and cortical tissue were previously injected with bromo-deoxyuridine (BrdU) on embryonic days 13 and 14 in order to label striatal neurons destined to populate the patch compartment of the striatum. Patches of BrdU-immunoreactive cells were maintained in organotypic cultures of late prenatal (embryonic days 20-22) or early postnatal striatum in the absence of nigral dopaminergic or cortical glutamatergic influences. In slices taken from embryonic day 19 fetuses prior to the time of in vivo patch formation, patches were observed to form after 10 days in vitro, in 39% of nigral-striatal co-cultures compared to 6% of striatal slices cultured alone or in the presence of cortex only. Patches of dopaminergic fibers, revealed by tyrosine hydroxylase immunoreactivity, were observed in the majority of nigral-striatal co-cultures. Immunostaining for the AMPA-type glutamate receptor GluR1 revealed a dense patch distribution in nearly all cultures, which developed in embryonic day 19 cultures after at least six days in vitro. These findings indicate that striatal patch/matrix organization is maintained in organotypic culture, and can be induced to form in vitro in striatal slices removed from fetuses prior to the time of in vivo patch formation. Furthermore, dopaminergic innervation from co-cultured pieces of ventral mesencephalon enhances patch

/sup 125/I-labelled tetanus toxin as a neuronal marker in tissue cultures derived from embryonic CNS

Energy Technology Data Exchange (ETDEWEB)

Dimpfel, W; Neale, J H; Habermann, E [Giessen Univ. (F.R. Germany). Pharmakologisches Inst.; National Inst. of Child Health and Human Development, Bethesda, Md. (USA). Behavioural Biology Branch)

1975-01-01

Primary cultures derived from embryonic mouse brain and spinal cord were exposed to /sup 125/I-labelled tetanus toxin and subjected to autoradioraphy. Cells with neuronal, but not glial, morphology selectively accumulated the toxin. The distribution of the grains over these cells and their processes was not uniform, discrete processes showing heavier labelling.

Effects of gentiopicroside, sweroside and swertiamarine, secoiridoids from gentian (Gentiana lutea ssp. symphyandra), on cultured chicken embryonic fibroblasts.

Science.gov (United States)

Oztürk, Nilgün; Korkmaz, Seval; Oztürk, Yusuf; Başer, K Hüsnü Can

2006-03-01

Wound healing properties of Gentian (Gentiana lutea ssp. symphyandra) extract and its main constituents, gentiopicroside, sweroside and swertiamarine (compounds 1-3, respectively) were evaluated by comparison with dexpanthenol on cultured chicken embryonic fibroblasts. The extract was also analyzed by HPLC to quantify its constituents. Chicken embryonic fibroblasts from fertilized eggs were incubated with the plant extract and its constituents, compounds 1-3. Using microscopy, mitotic ability, morphological changes and collagen production in the cultured fibroblasts were evaluated as parameters. Wound healing activity of Gentian seems to be mainly due to the increase in the stimulation of collagen production and the mitotic activity by compounds 2 and 3, respectively (p < 0.005 in all cases). All three compounds also exhibited cytoprotective effects, which may cause a synergism in terms of wound healing activity of Gentian. The findings demonstrated the wound healing activity of Gentian, which has previously been based only on ethnomedical data.

Generation of hematopoietic lineage cells from embryonic like cells

Directory of Open Access Journals (Sweden)

Gholam Reza Khamisipour

2014-10-01

Full Text Available Background: Epigenetic reprogramming of somatic cells into embryonic stem cells has attracted much attention, because of the potential for stem cell transplantation and compatibility with recipient. However, the therapeutic application of either nuclear transfer or nuclear fusion of somatic cell has been hindered by technical complications as well as ethical objections. Recently, a new method is reported whereby ectopic expression of embryonic specific transcription factors was shown to induce fibroblasts to become embryonic like SCs (induced pluripotent stem cells. A major limitation of this method is the use of potentially harmful genome integrating viruses such as reto- or lentivirus. The main aim of this investigation was generation of human hematopoietic stem cells from induced fibroblasts by safe adenovectors carrying embryonically active genes. Material and Methods: Isolated fibroblasts from foreskin were expanded and recombinant adenoviruses carrying human Sox2, Oct4, Klf4, cMyc genes were added to culture. After formation of embryonic like colonies and cell expansion, they were transferred to embryonic media without bFGF, and embryoid bodies were cultured on stromal and non-stromal differentiation media for 14 days. Results: Expression of CD34 gene and antigenic markers, CD34, CD38 & CD133 in stromal culture showed significant difference with non-differentiation and non-stromal media. Conclusion: These findings show high hematopoietic differentiation rate of Adeno-iPS cells in stromal culture and no need to use growth factors. While, there was no difference between non-differentiation and non-stromal media.

Embryonic mortality and intrauterine growth retardation (IUGR) associated with placental alterations in pregnant rats treated with methyl methanesulfonate (MMS) at the peri-implantation stage.

Science.gov (United States)

Yokoi, Ryohei; Hayashi, Morimichi; Tamura, Toru; Kobayashi, Kazuo; Kuroda, Junji; Kusama, Hiroshi; Kagami, Hiroshi; Ono, Tamao

2008-12-01

Embryonic mortality and intrauterine growth retardation (IUGR) are induced by exposure of rodents to xenobiotic agents during the pregastrulation period of development. We examined the time course of the effects of methyl methanesulfonate (MMS), an alkylating agent, on conceptus development in order to clarify the relative roles of the embryo and the placenta in their induction. Pregnant rats were treated orally with a single dose of MMS (200 mg/kg) in the morning of gestation day (GD) 6 (peri-implantation stage). Embryonic mortality was increased on GD12 and thereafter by MMS treatment, with newly dead embryos showing placental hypoplasia at GD12. Embryo or fetal weight was also smaller for MMS-treated dams than for control dams from GD14 to GD20. The labyrinth zone and junctional zone (JZ) of the placenta were thinner in MMS-treated rats from GD12 to GD17 and from GD12 to GD20 (except for GD17), respectively. Furthermore, MMS-treated dams showed a smaller number of glycogen cells in the JZ on GD14. In contrast, the placental glycogen concentration was higher and the expression of glucose transporter 1 in the JZ remained at GD20. These results indicate that exposure of pregnant rats to MMS at the peri-implantation stage of embryogenesis affects placental development and growth. The placental impairment induced by MMS was likely responsible for the embryonic death observed 6 days after exposure of dams to this agent as well as for the IUGR of surviving embryos or fetuses throughout the gestation period.

Benzodiazepine receptor turnover in embryonic chick brain and spinal cord cell cultures

International Nuclear Information System (INIS)

Borden, L.A.

1985-01-01

The turnover (synthesis and degradation) of the benzodiazepine receptor (BZD-R) in embryonic chick brain and spinal cord cell cultures was monitored using flunitrazepam (GNZM) as a photoaffinity label. To measure BZD-R appearance, intact cell cultures were incubated with 100 nM RNZM and irradiated with ultraviolet light; this process, referred to as photoinactivation, resulted in a 75% decrease in the subsequent reversible binding of 5 nM [ 3 H]FNZM. Following photoinactivation, [ 3 H]FNZM binding sites reappeared at a rate of 6 +/- 1.5%/hour (n = 7) in brain cultures and at 8%/hour (n = 2) in spinal cord cultures. Reappearance reflects de novo receptors synthesis. To examine the degradation of existing receptors, cultures were photolabeled with 5 nM [ 3 H]FNZM, washed, and then the decrease in cell-associated radioactivity, or the efflux of radioactivity into the medium, was monitored. The released radioactivity did not comigrate with authentic FNZM on thin-layer-chromatographs, indicating that release did not represent dissociation of ligand from the photolabeled receptor. The BZD-R appears to be degraded by an energy-dependent, non-lysosomal pathway. These experiments represent the first direct examination of the turnover of a neurotransmitter receptor localized to the central nervous system; this information will be valuable in elucidating the mechanisms by which receptor levels are altered following chronic drug treatment

Embryotoxic and pharmacologic potency ranking of six azoles in the rat whole embryo culture by morphological and transcriptomic analysis

International Nuclear Information System (INIS)

Dimopoulou, Myrto; Verhoef, Aart; Pennings, Jeroen L.A.; Ravenzwaay, Bennard van; Rietjens, Ivonne M.C.M.; Piersma, Aldert H.

2017-01-01

Differential gene expression analysis in the rat whole embryo culture (WEC) assay provides mechanistic insight into the embryotoxicity of test compounds. In our study, we hypothesized that comparative analysis of the transcriptomes of rat embryos exposed to six azoles (flusilazole, triadimefon, ketoconazole, miconazole, difenoconazole and prothioconazole) could lead to a better mechanism-based understanding of their embryotoxicity and pharmacological action. For evaluating embryotoxicity, we applied the total morphological scoring system (TMS) in embryos exposed for 48 h. The compounds tested showed embryotoxicity in a dose-response fashion. Functional analysis of differential gene expression after 4 h exposure at the ID 10 (effective dose for 10% decreased TMS), revealed the sterol biosynthesis pathway and embryonic development genes, dominated by genes in the retinoic acid (RA) pathway, albeit in a differential way. Flusilazole, ketoconazole and triadimefon were the most potent compounds affecting the RA pathway, while in terms of regulation of sterol function, difenoconazole and ketoconazole showed the most pronounced effects. Dose-dependent analysis of the effects of flusilazole revealed that the RA pathway related genes were already differentially expressed at low dose levels while the sterol pathway showed strong regulation at higher embryotoxic doses, suggesting that this pathway is less predictive for the observed embryotoxicity. A similar analysis at the 24-hour time point indicated an additional time-dependent difference in the aforementioned pathways regulated by flusilazole. In summary, the rat WEC assay in combination with transcriptomics could add a mechanistic insight into the embryotoxic potency ranking and pharmacological mode of action of the tested compounds. - Highlights: • Embryonic exposure to azoles revealed concentration-dependent malformations. • Transcriptomics could enhance the mechanistic knowledge of embryotoxicants. • Retinoic

Embryotoxic and pharmacologic potency ranking of six azoles in the rat whole embryo culture by morphological and transcriptomic analysis

Energy Technology Data Exchange (ETDEWEB)

Dimopoulou, Myrto, E-mail: myrto.dimopoulou@wur.nl [Division of Toxicology, Wageningen University (Netherlands); National Institute of Public Health and the Environment (RIVM), Bilthoven (Netherlands); Verhoef, Aart; Pennings, Jeroen L.A. [National Institute of Public Health and the Environment (RIVM), Bilthoven (Netherlands); Ravenzwaay, Bennard van [Division of Toxicology, Wageningen University (Netherlands); BASF SE, Experimental Toxicology and Ecology, Ludwigshafen (Germany); Rietjens, Ivonne M.C.M. [Division of Toxicology, Wageningen University (Netherlands); Piersma, Aldert H. [National Institute of Public Health and the Environment (RIVM), Bilthoven (Netherlands); Institute for Risk Assessment Sciences, Utrecht University, Utrecht (Netherlands)

2017-05-01

Differential gene expression analysis in the rat whole embryo culture (WEC) assay provides mechanistic insight into the embryotoxicity of test compounds. In our study, we hypothesized that comparative analysis of the transcriptomes of rat embryos exposed to six azoles (flusilazole, triadimefon, ketoconazole, miconazole, difenoconazole and prothioconazole) could lead to a better mechanism-based understanding of their embryotoxicity and pharmacological action. For evaluating embryotoxicity, we applied the total morphological scoring system (TMS) in embryos exposed for 48 h. The compounds tested showed embryotoxicity in a dose-response fashion. Functional analysis of differential gene expression after 4 h exposure at the ID{sub 10} (effective dose for 10% decreased TMS), revealed the sterol biosynthesis pathway and embryonic development genes, dominated by genes in the retinoic acid (RA) pathway, albeit in a differential way. Flusilazole, ketoconazole and triadimefon were the most potent compounds affecting the RA pathway, while in terms of regulation of sterol function, difenoconazole and ketoconazole showed the most pronounced effects. Dose-dependent analysis of the effects of flusilazole revealed that the RA pathway related genes were already differentially expressed at low dose levels while the sterol pathway showed strong regulation at higher embryotoxic doses, suggesting that this pathway is less predictive for the observed embryotoxicity. A similar analysis at the 24-hour time point indicated an additional time-dependent difference in the aforementioned pathways regulated by flusilazole. In summary, the rat WEC assay in combination with transcriptomics could add a mechanistic insight into the embryotoxic potency ranking and pharmacological mode of action of the tested compounds. - Highlights: • Embryonic exposure to azoles revealed concentration-dependent malformations. • Transcriptomics could enhance the mechanistic knowledge of embryotoxicants. �

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

OpenAIRE

Hunt, Geoffrey C.; Singh, Purva; Schwarzbauer, Jean E.

2012-01-01

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomita...

Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

Science.gov (United States)

Yamashita, Tomoko; Miyamoto, Yuki; Bando, Yoshio; Ono, Takashi; Kobayashi, Sakurako; Doi, Ayano; Araki, Toshihiro; Kato, Yosuke; Shirakawa, Takayuki; Suzuki, Yutaka; Yamauchi, Junji; Yoshida, Shigetaka; Sato, Naoya

2017-01-01

Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a

Simple explant culture of the embryonic chicken retina with long-term preservation of photoreceptors.

Science.gov (United States)

Thangaraj, Gopenath; Greif, Alexander; Layer, Paul G

2011-10-01

Structurally stable in vitro-model systems are indispensible to analyse neural development during embryogenesis, follow cellular differentiation and evaluate neurotoxicological or growth factor effects. Here we describe a three-dimensional, long-term in vitro-culture system of the embryonic chick retina which supports photoreceptor development. Retinal tissue was isolated from E6 chick eye, and cultured as explants by continuous orbital rotation to allow free floatation without any supporting materials. Young stage (E6) immature retinas were cultured for various time periods in order to follow the differentiation of cell types and plexiform layers by immunocytochemical methods. These explants could be cultured for at least 2-3 weeks with remarkable retention of retinal architecture. Interestingly, photoreceptors developed in the absence of pigment epithelium. Electron microscopic studies revealed formation of structures resembling photoreceptor outer segments, a feature not reported previously. Thus, the verification of photoreceptors, Müller cells, inner retinal cells and the inner plexiform layer described in our study establishes this explant culture as a valuable in vivo-like model system. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

A single-cell and feeder-free culture system for monkey embryonic stem cells.

Science.gov (United States)

Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

2014-01-01

Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

Stimulation of DNA synthesis in cultured rat alveolar type II cells

International Nuclear Information System (INIS)

Leslie, C.C.; McCormick-Shannon, K.; Robinson, P.C.; Mason, R.J.

1985-01-01

Restoration of the alveolar epithelium after injury is thought to be dependent on the proliferation of alveolar type II cells. To understand the factors that may be involved in promoting type II cell proliferation in vivo, we determined the effect of potential mitogens and culture substrata on DNA synthesis in rat alveolar type II cells in primary culture. Type II cells cultured in basal medium containing 10% fetal bovine serum (FBS) exhibited essentially no DNA synthesis. Factors that stimulated 3 H-thymidine incorporation included cholera toxin, epidermal growth factor, and rat serum. The greatest degree of stimulation was achieved by plating type II cells on an extracellular matrix prepared from bovine corneal endothelial cells and then by culturing the pneumocytes in medium containing rat serum, cholera toxin, insulin, and epidermal growth factor. Under conditions of stimulation of 3 H-thymidine incorporation there was an increased DNA content per culture dish but no increase in cell number. The ability of various culture conditions to promote DNA synthesis in type II cells was verified by autoradiography. Type II cells were identified by the presence of cytoplasmic inclusions, which were visualized by tannic acid staining before autoradiography. These results demonstrate the importance of soluble factors and culture substratum in stimulating DNA synthesis in rat alveolar type II cells in primary culture

Effects of Ethanol on the Expression Level of Various BDNF mRNA Isoforms and Their Encoded Protein in the Hippocampus of Adult and Embryonic Rats

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Shahla Shojaei

2015-12-01

Full Text Available We aimed to compare the effects of oral ethanol (Eth alone or combined with the phytoestrogen resveratrol (Rsv on the expression of various brain-derived neurotrophic factor (BDNF transcripts and the encoded protein pro-BDNF in the hippocampus of pregnant and embryonic rats. A low (0.25 g/kg body weight (BW/day dose of Eth produced an increase in the expression of BDNF exons I, III and IV and a decrease in that of the exon IX in embryos, but failed to affect BDNF transcript and pro-BDNF protein expression in adults. However, co-administration of Eth 0.25 g/kg·BW/day and Rsv led to increased expression of BDNF exons I, III and IV and to a small but significant increase in the level of pro-BDNF protein in maternal rats. A high (2.5 g/kg·BW/day dose of Eth increased the expression of BDNF exons III and IV in embryos, but it decreased the expression of exon IX containing BDNF mRNAs in the maternal rats. While the high dose of Eth alone reduced the level of pro-BDNF in adults, it failed to change the levels of pro-BDNF in embryos. Eth differentially affects the expression pattern of BDNF transcripts and levels of pro-BDNF in the hippocampus of both adult and embryonic rats.

ALTERATIONS IN THE DEVELOPING TESTIS TRANSCRIPTOME FOLLOWING EMBRYONIC VINCLOZOLIN EXPOSURE

OpenAIRE

Clement, Tracy M.; Savenkova, Marina I.; Settles, Matthew; Anway, Matthew D.; Skinner, Michael K.

2010-01-01

The current study investigates the direct effects of in utero vinclozolin exposure on the developing F1 generation rat testis transcriptome. Previous studies have demonstrated that exposure to vinclozolin during embryonic gonadal sex determination induces epigenetic modifications of the germ line and transgenerational adult onset disease states. Microarray analyses were performed to compare control and vinclozolin treated testis transcriptomes at embryonic day 13, 14 and 16. A total of 576 di...

The effects of low dose ionizing radiation on the development of rat cerebral cortex, (2); In vitro study with regards to neuronal migration

Energy Technology Data Exchange (ETDEWEB)

Matsushita, Koji [Kyoto Prefectural Univ. of Medicine (Japan)

1993-03-01

In order to study the molecular mechanisms of neuronal migration on developing rat cerebral cortex, we need a tissue culture system in which neuronal migration can be observed. We prepared a tissue culture system of embryonic rat cerebral cortex starting on embryonic day 16 and cultivating it for 48 hours. The autoradiographic study in this system revealed not only the migration of [sup 3]H-thymidine labeled neurons but also neuronal migration delays from low doses of ionizing radiation of more than 10 cGy. In addition, on immunohistochemical study, cell-cell adhesion molecule N-CAM staining was remarkably decreased in the matrix cell layer. In the tissue culture system where monoclonal anti-N-CAM antibodies were added, neuronal migration delay comparable to that of 20 cGy radiation was found. In conclusion, it was speculated that neuronal migration delay might be caused by disturbed N-CAM synthesis in matrix cells after low dose ionizing radiation. (author).

TOPICAL REVIEW: Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials

Science.gov (United States)

Amranul Haque, Md; Nagaoka, Masato; Hexig, Bayar; Akaike, Toshihiro

2010-02-01

Nanobiomaterials can play a central role in regenerative medicine and tissue engineering by facilitating cellular behavior and function, such as those where extracellular matrices (ECMs) direct embryonic stem (ES) cell morphogenesis, proliferation, differentiation and apoptosis. However, controlling ES cell proliferation and differentiation using matrices from natural sources is still challenging due to complex and heterogeneous culture conditions. Moreover, the systemic investigation of the regulation of self-renewal and differentiation to lineage specific cells depends on the use of defined and stress-free culture conditions. Both goals can be achieved by the development of biomaterial design targeting ECM or growth factors for ES cell culture. This targeted application will benefit from expansion of ES cells for transplantation, as well as the production of a specific differentiated cell type either by controlling the differentiation in a very specific pathway or by elimination of undesirable cell types.

Transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into rat spinal cord injuries does not cause harm.

Science.gov (United States)

Cloutier, Frank; Siegenthaler, Monica M; Nistor, Gabriel; Keirstead, Hans S

2006-07-01

Demyelination contributes to loss of function following spinal cord injury. We have shown previously that transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into adult rat 200 kD contusive spinal cord injury sites enhances remyelination and promotes recovery of motor function. Previous studies using oligodendrocyte lineage cells have noted a correlation between the presence of demyelinating pathology and the survival and migration rate of the transplanted cells. The present study compared the survival and migration of human embryonic stem cell-derived oligodendrocyte progenitors injected 7 days after a 200 or 50 kD contusive spinal cord injury, as well as the locomotor outcome of transplantation. Our findings indicate that a 200 kD spinal cord injury induces extensive demyelination, whereas a 50 kD spinal cord injury induces no detectable demyelination. Cells transplanted into the 200 kD injury group survived, migrated, and resulted in robust remyelination, replicating our previous studies. In contrast, cells transplanted into the 50 kD injury group survived, exhibited limited migration, and failed to induce remyelination as demyelination in this injury group was absent. Animals that received a 50 kD injury displayed only a transient decline in locomotor function as a result of the injury. Importantly, human embryonic stem cell-derived oligodendrocyte progenitor transplants into the 50 kD injury group did not cause a further decline in locomotion. Our studies highlight the importance of a demyelinating pathology as a prerequisite for the function of transplanted myelinogenic cells. In addition, our results indicate that transplantation of human embryonic stem cell-derived oligodendrocyte progenitor cells into the injured spinal cord is not associated with a decline in locomotor function.

Induction of osteogenic markers in differentially treated cultures of embryonic stem cells

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Ommerborn Michelle A

2008-06-01

Full Text Available Abstract Background Facial trauma or tumor surgery in the head and face area often lead to massive destruction of the facial skeleton. Cell-based bone reconstruction therapies promise to offer new therapeutic opportunities for the repair of bone damaged by disease or injury. Currently, embryonic stem cells (ESCs are discussed to be a potential cell source for bone tissue engineering. The purpose of this study was to investigate various supplements in culture media with respect to the induction of osteogenic differentiation. Methods Murine ESCs were cultured in the presence of LIF (leukemia inhibitory factor, DAG (dexamethasone, ascorbic acid and β-glycerophosphate or bone morphogenetic protein-2 (BMP-2. Microscopical analyses were performed using von Kossa staining, and expression of osteogenic marker genes was determined by real time PCR. Results ESCs cultured with DAG showed by far the largest deposition of calcium phosphate-containing minerals. Starting at day 9 of culture, a strong increase in collagen I mRNA expression was detected in the DAG-treated cells. In BMP-2-treated ESCs the collagen I mRNA induction was less increased. Expression of osteocalcin, a highly specific marker for osteogentic differentiation, showed a double-peaked curve in DAG-treated cells. ESCs cultured in the presence of DAG showed a strong increase in osteocalcin mRNA at day 9 followed by a second peak starting at day 17. Conclusion Supplementation of ESC cell cultures with DAG is effective in inducing osteogenic differentiation and appears to be more potent than stimulation with BMP-2 alone. Thus, DAG treatment can be recommended for generating ESC populations with osteogenic differentiation that are intended for use in bone tissue engineering.

Change of mitotic cycle and DNA repair in embryonic cells of rat, immortalized by E1 A oncogene and transformated by E1 A and c-Ha-Ras oncogenes under ionizing radiation action

International Nuclear Information System (INIS)

Kirillova, T.V.

1997-01-01

Comparison investigation into the repair of mitotic cycle and the reunion of DN single- and double-strand breaks in gamma-ray irradiated initial E1 A oncogene immortalized and E1 A and c-Ha-Ras oncogene transformed (mutant form) lines of rat embryonic fibroblasts was carried out. Possible involvement of Ras gene product in DNA repair speed governing and absence of tumor suppression function of p 53 protein in the embryonic and E1 A oncogene immortalized cells of rat fibroblast, as well as, presence of the mentioned function of p 53 protein in E1 A and c-Ha-Ras oncogene transformed cells were studied [ru

Three-dimensional co-culture facilitates the differentiation of embryonic stem cells into mature cardiomyocytes.

Science.gov (United States)

Ou, Dong-Bo; He, Yong; Chen, Rui; Teng, Ji-Wei; Wang, Hong-Tao; Zeng, Di; Liu, Xiong-Tao; Ding, Lu; Huang, Jin-Yan; Zheng, Qiang-Sun

2011-12-01

The cardiomyocyte (CM) differentiation of embryonic stem cells (ESCs) is routinely cultured as two-dimensional (2D) monolayer, which doesn't mimic in vivo physiological environment and may lead to low differentiated level of ESCs. Here, we develop a novel strategy that enhances CM differentiation of ESCs in collagen matrix three-dimensional (3D) culture combined with indirect cardiac fibroblasts co-culture. ESCs were cultured in hanging drops to form embryoid bodies (EBs) and then applied on collagen matrix. The EBs were indirectly co-cultured with cardiac fibroblasts by the hanging cell culture inserts (PET 1 µm). The molecular expressions and ultrastructural characteristics of ESC-derived CMs (ESCMs) were analyzed by real time RT-PCR, immunocytochemistry, and Transmission Electron Microscopy (TEM). We found that the percentage of beating EBs with cardiac fibroblasts co-culture was significantly higher than that without co-culture after differentiation period of 8 days. Type I collagen used as 3D substrates enhanced the late-stage CM differentiation of ESCs and had effect on ultrastructural mature of ESCMs in late-stage development. The combined effects of 3D and co-culture that mimic in vivo physiological environment further improved the efficiency of CM differentiation from ESCs, resulting in fiber-like structures of cardiac cells with organized sarcomeric structure in ESCMs. This novel 3D co-culture system emphasizes the fact that the ESC differentiation is actively responding to cues from their environment and those cues can drive phenotypic control, which provides a useful in vitro model to investigate CM differentiation of stem cells. Copyright © 2011 Wiley Periodicals, Inc.

Differentiation of Mouse Embryonic Stem Cells into Ventral Foregut Precursors

DEFF Research Database (Denmark)

Rothová, Michaela; Hölzenspies, Jurriaan J; Livigni, Alessandra

2016-01-01

Anterior definitive endoderm (ADE), the ventral foregut precursor, is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other organs. Here, a method is described for the differentiation of mouse embryonic stem cells (mESCs) to definitive...... endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (embryoid body) culture or in a serum-free adherent monolayer culture. ESC-derived ADE cells are committed to endodermal fates and can undergo further differentiation in vitro towards ventral foregut...

Affect of Bioglass {sup trademark} repeat dosage on mineralisation of embryonic bone 'in vitro'

Energy Technology Data Exchange (ETDEWEB)

Maroothynaden, J. [Imperial Coll. of Medicine, London (United Kingdom). Microgravity Tissue Engineering Lab.; Hench, L.L. [Imperial Coll. of Science, Technology and Medicine, London (United Kingdom). Dept. of Materials

2001-07-01

Utilising 45S5 Bioglass {sup trademark} extracts, as described previously, 16-day gestation embryonic mouse long-bones were cultured for 4-days while exposed to the same Bioglass{sup circledR} soluble extract solution for two different exposure times. In the first culture, all embryonic femurs were exposed to fresh 45S5 Bioglass {sup trademark} extract every 98 hours. In the second, the long-bones were exposed to fresh 45S5 Bioglass {sup trademark} extract solution every 48 hours. A simultaneous control culture was performed. All embryonic long-bone cultures mineralised after 4-days culture. Increasing the frequency of 45S5 Bioglass {sup trademark} exposure, from one exposure every 96 hrs to fresh exposures every 48 hrs, significantly increased the length and mineral content of the embryonic long-bones. (orig.)

Cultivo in vitro de eixos embrionários de paricá In vitro culture of paricá embryonic axes

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Iulla Naiff Rabelo de Souza Reis

2009-02-01

Full Text Available Objetivou-se, neste trabalho, avaliar a influência do meio de cultura MS normal e com as concentrações dos sais reduzidas à metade (½ MS, a presença de ácido giberélico (AG3 e de ácido cítrico em eixos embrionários de paricá cultivados in vitro. Os eixos embrionários foram extraídos das sementes e inoculados em meio de cultura básico MS e ½ MS, suplementados ou não com ácido cítrico (1 g.L-1 e com AG3 (3 mg.L-1, constituindo 6 tratamentos e 7 repetições. Os eixos embrionários de paricá podem ser cultivados em meio MS com metade das concentrações dos sais, não sendo necessária a adição de ácido cítrico, e a presença de 3 mg.L-1 de AG3 afetou o desenvolvimento das plântulas de paricá.The aim of this study was to evaluate the influence of normal MS culture medium and at half strength of its salts (½ MS, the presence of gibberellic acid (GA3 and citric acid on the in vitro culture of paricá embryonic axes. The embryonic axes were extracted from seeds and inoculated on basic MS and ½ MS culture media, supplemented or not with citric acid (1 g.L-1 and GA3 (3 mg.L-1, constituting 6 treatments and 7 replications. The embryonic axes of paricá may be cultivated on MS medium at half strength of its salts, the addition of citric acid not being necessary, and the presence of 3 mg.L-1 GA3 affected the development of paricá seedling.

Promotion of hair follicle development and trichogenesis by Wnt-10b in cultured embryonic skin and in reconstituted skin

International Nuclear Information System (INIS)

Ouji, Yukiteru; Yoshikawa, Masahide; Shiroi, Akira; Ishizaka, Shigeaki

2006-01-01

We previously showed that Wnt-10b promoted the differentiation of primary skin epithelial cells (MPSEC) toward hair shaft and inner root sheath of the hair follicle (IRS) cells in vitro. In the present study, we found that Wnt-10b promotes the development of hair follicles using a culture of mouse embryonic skin tissue and trichogenesis using a reconstitution experiment with nude mice. Hair follicle development was observed in skin taken from mouse embryos on embryonic day 10.5 following a 2-day culture with recombinant Wnt-10b (rWnt-10b), however, not without rWnt-10b. Brown hair growth was observed at the site of reconstituted skin in Balb/c nude mice where dermal fibroblasts and keratinocytes, derived from C3H/HeN new born mice, were transplanted with Wnt-10b-producing COS cells (Wnt-COS). Without the co-transplantation of Wnt-COS, no hair growth was observed. Our results suggest an important role of Wnt-10b in the initiation of hair follicle development and following trichogenesis

Higher-Density Culture in Human Embryonic Stem Cells Results in DNA Damage and Genome Instability

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Kurt Jacobs

2016-03-01

Full Text Available Human embryonic stem cells (hESC show great promise for clinical and research applications, but their well-known proneness to genomic instability hampers the development to their full potential. Here, we demonstrate that medium acidification linked to culture density is the main cause of DNA damage and genomic alterations in hESC grown on feeder layers, and this even in the short time span of a single passage. In line with this, we show that increasing the frequency of the medium refreshments minimizes the levels of DNA damage and genetic instability. Also, we show that cells cultured on laminin-521 do not present this increase in DNA damage when grown at high density, although the (long-term impact on their genomic stability remains to be elucidated. Our results explain the high levels of genome instability observed over the years by many laboratories worldwide, and show that the development of optimal culture conditions is key to solving this problem.

Impact of electromagnetic radiation exposure during pregnancy on embryonic skeletal development in rats

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Ali SAEED H Alchalabi

2017-03-01

Full Text Available Objective: To evaluate the teratogenic effect of mobile phone radiation exposure during pregnancy on embryonic skeletal development at the common used mobile phone frequency in our environment. Methods: Sixty female Sprague-Dawley rats were distributed into three experiment groups; control and two exposed groups (1 h/day, 2 h/day exposure groups (n=20/ each group and exposed to whole body radiation during gestation period from day 1- day 20. Electromagnetic radiofrequency signal generator was used to generate 1 800 MHz GSM-like signals at specific absorption rate value 0.974 W/kg. Animals were exposed during experiment in an especial designed Plexiglas box (60 cm × 40 cm × 30 cm. At the end of exposure duration at day 20 of pregnancy animals were sacrificed and foetuses were removed, washed with normal saline and processed to Alizarin red and Alcian blue stain. Skeleton specimens were examined under a stereo microscope and skeleton's snaps were being carefully captured by built in camera fixed on the stereo microscope. Results: Intrauterine exposure to electromagnetic radiation lead to variation in degree of ossification, mineralization, formation of certain parts of the skeleton majorly in head and lesser in other parts. Deformity and absence of formation of certain bones in the head, ribs, and coccygeal vertebrae were recorded in skeleton of foetuses from exposed dams compare to control group. Conclusions: The electromagnetic radiation exposure during pregnancy alter the processes of bone mineralization and the intensity of bone turnover processes, and thus impact embryonic skeleton formation and development directly.

Rat embryonic fibroblasts improve reprogramming of human keratinocytes into induced pluripotent stem cells.

Science.gov (United States)

Linta, Leonhard; Stockmann, Marianne; Kleinhans, Karin N; Böckers, Anja; Storch, Alexander; Zaehres, Holm; Lin, Qiong; Barbi, Gotthold; Böckers, Tobias M; Kleger, Alexander; Liebau, Stefan

2012-04-10

Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is, however, limited and thereby further optimization in terms of time, efficiency, and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1, Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells. © Mary Ann Liebert, Inc.

Regulation of Taurine transporter activity in cultured rat retinal ganglion cells and rat retinal Muller Cells

International Nuclear Information System (INIS)

Eissa, Laila A.; Smith, Sylvia B.; El-sherbeny, Amira A.

2006-01-01

Diabetic retinopathy is one of the most common complications of diabetes. The amino acid taurine is believed to play an antioxidant protective role in diabetic retinopathy through the scavenging of the reactive species. It is not well established whether taurine uptake is altered in retina cells during diabetic conditions. Thus, the present study was designed to investigate the changes in taurine transport in cultures of rat retinal Muller cells and rat retinal ganglion cells under conditions associated with diabetes. Taurine was abundantly taken up by retinal Muller cells and rat retinal ganglion cells under normal glycemic condition. Taurine was actively transported to rat Muller cells and rat retinal ganglion cells in a Na and Cl dependant manner. Taurine uptake further significantly elevated in both type of cells after the incubation with high glucose concentration. This effect could be attributed to the increase in osmolarity. Because Nitric Oxide (NO) is a molecule implicated in the pathogenesis of diabetes, we also determined the activity of taurine transporter in cultured rat retinal Muller cells and rat retinal ganglion cells in the presence of the NO donors, SIN-1 and SNAP. Taurine uptake was elevated above control value after 24-h incubation with low concentration of NO donors. We finally investigated the ability of neurotoxic glutamate to change taurine transporter activity in both types of cells. Uptake of taurine was significantly increased in rat retinal ganglion cells when only incubated with high concentration of glutamate. Our data provide evidence that taurine transporter is present in cultured rat retinal ganglion and Muller cells and is regulated by hyperosmolarity. The data are relevant to disease such as diabetes and neuronal degeneration where retinal cell volume may dramatically change. (author)

Erythroid differentiation and commitment in rat erythroleukemia cells with hypertonic culture conditions.

OpenAIRE

Yamaguchi, Y; Kluge, N; Ostertag, W; Furusawa, M

1981-01-01

Cell cultures of 7,12-dimethylbenz[a]anthracene-induced rat erythroleukemia can be stimulated to synthesize hemoglobin when cultured in hypertonic media. During hypertonic treatment the intracellular osmotic conditions immediately readjust to those of the extracellular medium. None of the Friend virus-induced mouse erythroleukemia cell lines was inducible for differentiation with the same hypertonic culture conditions used for rat cells. Earliest commitment to erythroid terminal differentiati...

Electrochemical lactate biosensor based upon chitosan/carbon nanotubes modified screen-printed graphite electrodes for the determination of lactate in embryonic cell cultures.

Science.gov (United States)

Hernández-Ibáñez, Naiara; García-Cruz, Leticia; Montiel, Vicente; Foster, Christopher W; Banks, Craig E; Iniesta, Jesús

2016-03-15

l-lactate is an essential metabolite present in embryonic cell culture. Changes of this important metabolite during the growth of human embryo reflect the quality and viability of the embryo. In this study, we report a sensitive, stable, and easily manufactured electrochemical biosensor for the detection of lactate within embryonic cell cultures media. Screen-printed disposable electrodes are used as electrochemical sensing platforms for the miniaturization of the lactate biosensor. Chitosan/multi walled carbon nanotubes composite have been employed for the enzymatic immobilization of the lactate oxidase enzyme. This novel electrochemical lactate biosensor analytical efficacy is explored towards the sensing of lactate in model (buffer) solutions and is found to exhibit a linear response towards lactate over the concentration range of 30.4 and 243.9 µM in phosphate buffer solution, with a corresponding limit of detection (based on 3-sigma) of 22.6 µM and exhibits a sensitivity of 3417 ± 131 µAM(-1) according to the reproducibility study. These novel electrochemical lactate biosensors exhibit a high reproducibility, with a relative standard deviation of less than 3.8% and an enzymatic response over 82% after 5 months stored at 4 °C. Furthermore, high performance liquid chromatography technique has been utilized to independently validate the electrochemical lactate biosensor for the determination of lactate in a commercial embryonic cell culture medium providing excellent agreement between the two analytical protocols. Copyright © 2015 Elsevier B.V. All rights reserved.

The effects of 1α, 25-dihydroxyvitamin D3 and transforming growth factor-β3 on bone development in an ex vivo organotypic culture system of embryonic chick femora.

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Emma L Smith

Full Text Available Transforming growth factor-beta3 (TGF-β3 and 1α,25-dihydroxyvitamin D3 (1α,25 (OH 2D3 are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH2D3 and TGF-β3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11 were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH 2D3 (25 nM or TGF-β3 (5 ng/mL & 15 ng/mL. Analyses of the femurs were undertaken using micro-computed tomography (μCT, histology and immunohistochemistry. 1α,25 (OH2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH 2D3 and TGF-β3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH2D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.

Hypoxia preferentially destroys GABAergic neurons in developing rat neocortex explants in culture

NARCIS (Netherlands)

Romijn, H. J.; Ruijter, J. M.; Wolters, P. S.

1988-01-01

The hypothesis that hypoxic ischemia before or during the human birth process preferentially destroys GABAergic nerve cells, particularly in the neocortex, was tested in a tissue culture model system. To that end, rat neocortex explants dissected from 6-day-old rat pups and cultured to a

A dysmorphology score system for assessing embryo abnormalities in rat whole embryo culture.

Science.gov (United States)

Zhang, Cindy X; Danberry, Tracy; Jacobs, Mary Ann; Augustine-Rauch, Karen

2010-12-01

The rodent whole embryo culture (WEC) system is a well-established model for characterizing developmental toxicity of test compounds and conducting mechanistic studies. Laboratories have taken various approaches in describing type and severity of developmental findings of organogenesis-stage rodent embryos, but the Brown and Fabro morphological score system is commonly used as a quantitative approach. The associated score criteria is based upon developmental stage and growth parameters, where a series of embryonic structures are assessed and assigned respective scores relative to their gestational stage, with a Total Morphological Score (TMS) assigned to the embryo. This score system is beneficial because it assesses a series of stage-specific anatomical landmarks, facilitating harmonized evaluation across laboratories. Although the TMS provides a quantitative approach to assess growth and determine developmental delay, it is limited to its ability to identify and/or delineate subtle or structure-specific abnormalities. Because of this, the TMS may not be sufficiently sensitive for identifying compounds that induce structure or organ-selective effects. This study describes a distinct morphological score system called the "Dysmorphology Score System (DMS system)" that has been developed for assessing gestation day 11 (approximately 20-26 somite stage) rat embryos using numerical scores to differentiate normal from abnormal morphology and define the respective severity of dysmorphology of specific embryonic structures and organ systems. This method can also be used in scoring mouse embryos of the equivalent developmental stage. The DMS system enhances capabilities to rank-order compounds based upon teratogenic potency, conduct structure- relationships of chemicals, and develop statistical prediction models to support abbreviated developmental toxicity screens. © 2010 Wiley-Liss, Inc.

Effect of culture medium volume and embryo density on early mouse embryonic development: tracking the development of the individual embryo.

Science.gov (United States)

Dai, Shan-Jun; Xu, Chang-Long; Wang, Jeffrey; Sun, Ying-Pu; Chian, Ri-Cheng

2012-07-01

To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly. (1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining. The blastocyst development was significantly higher (P WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P WOW system than without. Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 μl of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced

Studies on the toxic effects of periodontal sustained release drug containing ornidazole and pefloxacin mesylate on early embryonic development of SD rat

Directory of Open Access Journals (Sweden)

Zheng-mou DONG

2011-01-01

Full Text Available Objective To evaluate the toxic effects of periodontal sustained release drug containing ornidazole and pefloxacin mesylate on early embryonic development of SD rats.Methods A total of 100female SD rats were randomly divided into negative control,low-,medium-,high-dose group and intervention group(20each.Rats in low-,medium-and high-dose group were fed daily with the sustained release drug at 1,4,and 8g/kg respectively;those in negative control group were fed daily with distilled water from the 14th day before mating to the 7th day of pregnancy continuously,and those in intervention group received cyclophosphamide(40mg/kgby intraperitoneal injection for 5successive days.During this period,the general status,mating,pregnancy,coefficient of ovary and uterus,the numbers of corpus luteum,nidation,live births,stillbirths,absorbed embryo,prenidatory and postnidatory mortality,serum testosterone(Tand estradiol(E2were determined respectively.Histopathologic examination of the ovary and uterus,immunohistochemical observation of ovaries for proliferating cell nuclear antigen(PCNAand Bcl-2associated X protein(Baxwere also performed respectively.Results The general status of those rats was good except one in the low-dose group and one in the intervention group died on the 14th day of administration,and one in negative control and one in high dose group died on the 5th day of pregnancy,respectively.The body weight of animals decreased significantly(P 0.05.The serum T level in medium-and high-dose group and the E2level in high-dose group declined compared to that in negative control group(P < 0.05.Conclusions Although the periodontal sustained release drug containing ornidazole and pefloxacin mesylate shows no toxicity to the early embryonic development of SD rats,the high dose drug has certain toxicity to ovary.Declined serum concentrations of T and E2,reduced expression of PCNA,and increased Bax may be the causes of the toxicity.

Physiological oxygen prevents frequent silencing of the DLK1-DIO3 cluster during human embryonic stem cells culture.

Science.gov (United States)

Xie, Pingyuan; Sun, Yi; Ouyang, Qi; Hu, Liang; Tan, Yueqiu; Zhou, Xiaoying; Xiong, Bo; Zhang, Qianjun; Yuan, Ding; Pan, Yi; Liu, Tiancheng; Liang, Ping; Lu, Guangxiu; Lin, Ge

2014-02-01

Genetic and epigenetic alterations are observed in long-term culture (>30 passages) of human embryonic stem cells (hESCs); however, little information is available in early cultures. Through a large-scale gene expression analysis between initial-passage hESCs (ihESCs, cell derivatives, possibly through attenuation of the expression and phosphorylation of p53. Furthermore, we demonstrated that 5% oxygen, instead of the commonly used 20% oxygen, is required for preserving the expression of the DLK1-DIO3 cluster. Overall, the data suggest that active expression of the DLK1-DIO3 cluster represents a new biomarker for epigenetic stability of hESCs and indicates the importance of using a proper physiological oxygen level during the derivation and culture of hESCs. © AlphaMed Press.

Cytoarchitecture in cultured rat neocortex explants

NARCIS (Netherlands)

de Jong, B. M.; Ruijter, J. M.; Romijn, H. J.

1988-01-01

Neocortex explants obtained from 6-day-old rat pups and cultured in a serum-free medium from 5 hr to 13 days in vitro (DIV) show preservation of cytoarchitectural characteristics. Major changes in the size of the explants and their layers occur during the first 2 DIV. A radial arrangement of neurons

Safflower and olive oil dietary treatments rescue aberrant embryonic arachidonic acid and nitric oxide metabolism and prevent diabetic embryopathy in rats.

Science.gov (United States)

Higa, R; White, V; Martínez, N; Kurtz, M; Capobianco, E; Jawerbaum, A

2010-04-01

Aberrant arachidonic acid and nitric oxide (NO) metabolic pathways are involved in diabetic embryopathy. Previous works have found diminished concentrations of PGE(2) and PGI(2) in embryos from diabetic rats, and that PGI(2) is capable of increasing embryonic PGE(2) concentrations through the activation of the nuclear receptor PPARdelta. PPARdelta activators are lipid molecules such as oleic and linoleic acids, present in high concentrations in olive and safflower oils, respectively. The aim of this study was to analyze the capability of dietary supplementation with either 6% olive or 6% safflower oils to regulate PGE(2), PGI(2) and NO concentrations in embryos and deciduas from control and diabetic rats during early organogenesis. Diabetes was induced by a single injection of streptozotocin (55 mg/kg) 1 week before mating. Animals were fed with the oil-supplemented diets from Days 0.5 to 10.5 of gestation. PGI(2) and PGE(2) were measured by EIA and NO through the evaluation of its stable metabolites nitrates-nitrites in 10.5 day embryos and deciduas. We found that the olive and safflower oil-supplemented treatments highly reduced resorption and malformation rates in diabetic animals, and that they were able to prevent maternal diabetes-induced alterations in embryonic and decidual PGI(2) and PGE(2) concentrations. Moreover, these dietary treatments prevented NO overproduction in embryos and deciduas from diabetic rats. These data indicate that in maternal diabetes both the embryo and the decidua benefit from the olive and safflower oil supplementation probably through mechanisms that involve the rescue of aberrant prostaglandin and NO generation and that prevent developmental damage during early organogenesis.

Differentiating Mouse Embryonic Stem Cells into Embryoid Bodies by Hanging-Drop Cultures.

Science.gov (United States)

Behringer, Richard; Gertsenstein, Marina; Nagy, Kristina Vintersten; Nagy, Andras

2016-12-01

Embryonic stem (ES) cells can develop into many types of differentiated tissues if they are placed into a differentiating environment. This can occur in vivo when the ES cells are injected into or aggregated with an embryo, or in vitro if their culture conditions are modified to induce differentiation. There are an increasing number of differentiating culture conditions that can bias the differentiation of ES cells into desired cell types. Determining the mechanisms that control ES cell differentiation into therapeutically important cell types is a quickly growing area of research. Knowledge gained from these studies may eventually lead to the use of stem cells to repair specific damaged tissues. Many times ES cell differentiation proceeds through an intermediate stage called the embryoid body (EB). EBs are round structures composed of ES cells that have undergone some of the initial stages of differentiation. EBs can then be manipulated further to generate more specific cell types. This protocol describes a method to differentiate ES cells into EBs. It produces EBs of comparable size. This aspect is important because the differentiation processes taking place inside an EB are influenced by its size. © 2016 Cold Spring Harbor Laboratory Press.

Distribution of epidermal growth factor receptors in rat tissues during embryonic skin development, hair formation, and the adult hair growth cycle

DEFF Research Database (Denmark)

Green, M R; Couchman, J R

1984-01-01

on the binding distribution of [125I]EGF, representing the tissue localization of available EGF receptors, during embryonic rat skin development including hair follicle formation and the adult hair growth cycle. At 16 days embryonic development a relatively low receptor density is seen over all the epidermal...... condensates marking the first stage of hair follicle development. This restricted and temporary loss of EGF receptors above these specialized mesenchymal condensates implies a role for the EGF receptor and possibly EGF or an EGF-like ligand in stimulating the epithelial downgrowth required for hair follicle...... development. In the anagen hair bulb, receptors for EGF are detected over the outer root sheath and the epithelial cell layers at the base of the follicle and show a correlation with the areas of epithelial proliferation in the hair bulb. During the catagen and telogen phases of the hair cycle, receptors...

Stable isotope labelling with amino acids in cell culture for human embryonic stem cell proteomic analysis

DEFF Research Database (Denmark)

Harkness, Linda; Prokhorova, Tatyana A; Kassem, Moustapha

2012-01-01

The identification and quantitative measurements of proteins in human embryonic stem cells (hESC) is a fast growing interdisciplinary area with an enormous impact on understanding the biology of hESC and the mechanism controlling self-renewal and differentiation. Using a quantitative mass...... spectroscopic method of stable isotope labelling with amino acids during cell culture (SILAC), we are able to analyse differential expression of proteins from different cellular compartments and to identify intracellular signalling pathways involved in self-renewal and differentiation. In this chapter, we...

Characterization of p75+ ectomesenchymal stem cells from rat embryonic facial process tissue

International Nuclear Information System (INIS)

Wen, Xiujie; Liu, Luchuan; Deng, Manjing; Zhang, Li; Liu, Rui; Xing, Yongjun; Zhou, Xia; Nie, Xin

2012-01-01

Highlights: ► Ectomesenchymal stem cells (EMSCs) were found to migrate to rat facial processes at E11.5. ► We successfully sorted p75NTR positive EMSCs (p75 + EMSCs). ► p75 + EMSCs up to nine passages showed relative stable proliferative activity. ► We examined the in vitro multilineage potential of p75 + EMSCs. ► p75 + EMSCs provide an in vitro model for tooth morphogenesis. -- Abstract: Several populations of stem cells, including those from the dental pulp and periodontal ligament, have been isolated from different parts of the tooth and periodontium. The characteristics of such stem cells have been reported as well. However, as a common progenitor of these cells, ectomesenchymal stem cells (EMSCs), derived from the cranial neural crest have yet to be fully characterized. The aim of this study was to better understand the characteristics of EMSCs isolated from rat embryonic facial processes. Immunohistochemical staining showed that EMSCs had migrated to rat facial processes at E11.5, while the absence of epithelial invagination or tooth-like epithelium suggested that any epithelial–mesenchymal interactions were limited at this stage. The p75 neurotrophin receptor (p75NTR), a typical neural crest marker, was used to select p75NTR-positive EMSCs (p75 + EMSCs), which were found to show a homogeneous fibroblast-like morphology and little change in the growth curve, proliferation capacity, and cell phenotype during cell passage. They also displayed the capacity to differentiate into diverse cell types under chemically defined conditions in vitro. p75 + EMSCs proved to be homogeneous, stable in vitro and potentially capable of multiple lineages, suggesting their potential for application in dental or orofacial tissue engineering.

In vitro culture and characterization of putative porcine embryonic germ cells derived from domestic breeds and yucatan mini pig embryos at days 20-24 of gestation

DEFF Research Database (Denmark)

Petkov, Stoyan Gueorguiev; Marks, Hendrik; Klein, Tino

2011-01-01

Embryonic germ cells (EGC) are cultured pluripotent cells derived from primordial germ cells (PGC). This study explored the possibility of establishing porcine EGC from domestic breeds and Yucatan mini pigs using embryos at Days 17-24 of gestation. In vitro culture of PGC from both pooled...

Endolymphatic potassium of the chicken vestibule during embryonic development.

Science.gov (United States)

Masetto, Sergio; Zucca, Giampiero; Bottà, Luisa; Valli, Paolo

2005-08-01

The endolymph fills the lumen of the inner ear membranous labyrinth. Its ionic composition is unique in vertebrates as an extracellular fluid for its high-K(+)/low-Na(+) concentration. The endolymph is actively secreted by specialized cells located in the vestibular and cochlear epithelia. We have investigated the early phases of endolymph secretion by measuring the endolymphatic K(+) concentration in the chicken vestibular system during pre-hatching development. Measurements were done by inserting K(+)-selective microelectrodes in chicken embryo ampullae dissected at different developmental stages from embryonic day 9 up to embryonic day 21 (day of hatching). We found that the K(+) concentration is low (<10mM/L) up to embryonic day 11, afterward it increases steeply to reach a plateau level of about 140 mM/L at embryonic day 19--21. We have developed a short-term in vitro model of endolymph secretion by culturing vestibular ampullae dissected from embryonic day 11 chicken embryos for a few days. The preparation reproduced a double compartment system where the luminal K(+) concentration increased along with the days of culturing. This model could be important for (1) investigating the development of cellular mechanisms contributing to endolymph homeostasis and (2) testing compounds that influence those mechanisms.

Isolation, culture and intraportal transplantation of rat marrow stromal cell

International Nuclear Information System (INIS)

Wang Ping; Wang Jianhua; Yan Zhiping; Li Wentao; Lin Genlai; Hu Meiyu; Wang Yanhong

2004-01-01

Objective: To observe the tracing and evolution of marrow stromal cell (MSC) after intraportal transplantation into the liver of homogenous rats, and to provide experimental data for MSC differentiation to hepatocyte in vivo. Methods: The MSC was isolated from the leg bone marrow of adult SD rats, and purified by culture-expanded in vitro. Before transplantation, MSC was labeled with DAPI. Then 10 5 MSC were intraportally transplanted into the homogenous rat liver. Rats were killed at 2 hours and 1, 2, 3 and 4 weeks after transplantation. The cryosection samples of liver and lung were observed under fluorescence microscopy. Results: MSC in vitro culture had high ability of proliferation. Except 4 rats were dead because of abdominal bleeding or infection, other recipients were healthy until sacrificed. The implantation cells were detected by identifying the DAPI labeled MSC in the host livers, but not in the host lungs. Conclusion: Intraportal transplanted MSC could immigrate and survive in the host livers at least for 4 weeks. They could immigrate from the small branches of portal veins to hepatic parenchyma

Degenerative changes and cell death in long-living homo- and heterotopic transplants from embryonic germ layers of rat neocortex.

Science.gov (United States)

Petrova, E S; Otellin, V A

2003-09-01

Morphological study of allotransplants of rat embryonic neocortex 14-18 months after transplantation into the neocortex, lateral cerebral ventricle, and sciatic nerve of adult animals revealed death of nerve and glial cells in the delayed postoperation period independently on the site of transplantation. After heterotopic transplantation the count of degenerated neurons was 2 times higher that after homotopic transplantation. In heterotopic transplants a considerable number of grafted neurons underwent reversible and irreversible degenerative changes accompanied by their premature aging. Neuronal death is probably determined by insufficiency of trophic influence from afferent structures and target tissues. We hypothesized that antiapoptotic preparations can be used for prevention of transplanted cell death. It was also found that degeneration of neurons was associated with impaired vascularization of transplants and pronounced immune reaction of the recipient in late posttransplantation period. Transplantation of embryonic brain structures can serve as a model system in studies concerning involutive and pathological processes in the central nervous system and in the search for factors improving survival of neurons.

Effects of Forskolin on Trefoil factor 1 expression in cultured ventral mesencephalic dopaminergic neurons

DEFF Research Database (Denmark)

Jensen, Pia; Ducray, A D; Widmer, H R

2015-01-01

shown that TFF1 is expressed in developing and adult rat ventral mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons. Here, we investigated the expression of TFF1 in rat ventral mesencephalic dopaminergic neurons (embryonic day 14) grown in culture for 5, 7 or 10days......, suggesting that Forskolin induced TFF1 expression through diverse signaling pathways. In conclusion, distinct populations of cultured dopaminergic neurons express TFF1, and their numbers can be increased by factors known to influence survival and differentiation of dopaminergic cells....... to neuronal cells, and the percentage of TH/TFF1 co-expressing cells was increased to the same extent in GDNF and Forskolin-treated cultures (4-fold) as compared to controls. Interestingly, the combination of GDNF and Forskolin resulted in a significantly increased co-expression (8-fold) of TH/TFF1, which...

A method for isolating identifying and culturing of rat trachea-bronchia epithelial cells

International Nuclear Information System (INIS)

Cui Fengmei; Su Shibiao; Nie Jihua; Li Bingyan; Tong Jian

2005-01-01

Objective: To explore a method for isolating identifying and culturing the rat trachea-bronchia epithelial cells. Methods: The rat trachea-bronchia epithelial cells were isolated by digestion with pronase and brushing with cell brush, identified using confocul and cultured in entire F12 media with no serum. Results: With this method, cells in high purity and high viability could be obtained, and about 10 6 cells per rat. The cells grow well in entire F12 media with no serum. Conclusion: The method is useful for isolating rate trachea-bronchia epithelial cells and the entire F12 media with no serum is effective for culturing. (authors)

Differentiation of cartilaginous anlage in entire embryonic mouse limbs cultured in a rotating bioreactor.

Science.gov (United States)

Duke, P.; Oakley, C.; Montufar-Solis, D.

The embryonic mammalian limb is sensitive both in vivo and in vitro to changes in gravitational force. Hypergravity of centrifugation and microgravity of space decreased size of elements due to precocious or delayed chondrogenesis respectively. In recapitulating spaceflight experiments, premetatarsals were cultured in suspension in a low stress, low sheer rotating bioreactor, and found to be shorter than those cultured in standard culture dishes, and cartilage development was delayed. This study only measured length of the metatarsals, and did not account for possible changes in width and/or in form of the skeletal elements. Shorter cartilage elements in limbbuds cultured in the bioreactor may be due to the ability of the system to reproduce a more in vivo 3D shape than traditional organ cultures. Tissues subjected to traditional organ cultures become flattened by their own weight, attachment to the filter, and restrictions imposed by nutrient diffusion. The purpose of the current experiment was to determine if entire limb buds could be successfully cultured in the bioreactor, and to compare the effects on 3D shape with that of culturing in a culture dish system. Fore and hind limbs from E11-E13 ICR mouse embryos were placed either in the bioreactor, in Trowell culture, or fixed as controls. Limbbuds were cultured for six days, fixed, and processed either as whole mounts or embedded for histology. Qualitative analysis revealed that the Trowell culture specimens were flattened, while bioreactor culture specimens had a more in vivo-like 3D limb shape. Sections of limbbuds from both types of cultures had excellent cartilage differentiation, with apparently more cell maturation, and hypertrophy in the specimens cultured in the bioreactor. Morphometric quantitation of the cartilaginous elements for comparisons of the two culture systems was complicated due to some limb buds fusing together during culture. This problem was especially noticeable in the younger limbs, and

Chorionic villi derived mesenchymal like stem cells and expression of embryonic stem cells markers during long-term culturing.

Science.gov (United States)

Katsiani, E; Garas, A; Skentou, C; Tsezou, A; Messini, C I; Dafopoulos, K; Daponte, A; Messinis, I E

2016-09-01

Mesenchymal stem cells (MSCs) can be obtained from a variety of human tissues. MSCs derived from placental chorionic villi of the first trimester are likely to resemble, biologically, embryonic stem cells (ESC), due to the earlier development stage of placenta. In the present study long-term cultures of MSC-like cells were assessed in order to evaluate MSCs multipotent characteristics and molecular features during the period of culture. CV-cells obtained from 10 samples of chorionic villus displayed typical fibroblastoid morphology, undergone 20 passages during a period of 120 days, maintaining a stable karyotype throughout long term expansion. The cells were positive, for CD90, CD73, CD105, CD29, CD44, HLA ABC antigens and negative for CD14, CD34, AC133, and HLA DR antigens as resulted from the flow cytometry analysis. CV-cells were differentiated in adipocytes, osteoblasts, chondrocytes and neuronal cells under specific culture conditions. The expression of the ESC-gene markers POU5F1 (Oct-4) and NANOG was observed at earliest stages (4-12 passages) and not at the late stages (14-20 passages) by RT-PCR analysis. ZFP42 and SOX2 expression were not detected. Moreover, CV-cells were found to express GATA4 but not NES (Nestin). Chorionic villi-derived cells possess multipotent properties, display high proliferation rate and self-renew capacity, share common surface antigens with adult MSCs and express certain embryonics stem cells gene markers. These characteristics highlight chorionic villi as an attractive source of MSCs for the needs of regenerative medicine.

Comparison of the mouse Embryonic Stem cell Test, the rat Whole Embryo Culture and the Zebrafish Embryotoxicity Test as alternative methods for developmental toxicity testing of six 1,2,4-triazoles

International Nuclear Information System (INIS)

Jong, Esther de; Barenys, Marta; Hermsen, Sanne A.B.; Verhoef, Aart; Ossendorp, Bernadette C.; Bessems, Jos G.M.; Piersma, Aldert H.

2011-01-01

The relatively high experimental animal use in developmental toxicity testing has stimulated the search for alternatives that are less animal intensive. Three widely studied alternative assays are the mouse Embryonic Stem cell Test (EST), the Zebrafish Embryotoxicity Test (ZET) and the rat postimplantation Whole Embryo Culture (WEC). The goal of this study was to determine their efficacy in assessing the relative developmental toxicity of six 1,2,4-triazole compounds, flusilazole, hexaconazole, cyproconazole, triadimefon, myclobutanil and triticonazole. For this purpose, we analyzed effects and relative potencies of the compounds in and among the alternative assays and compared the findings to their known in vivo developmental toxicity. Triazoles are antifungal agents used in agriculture and medicine, some of which are known to induce craniofacial and limb abnormalities in rodents. The WEC showed a general pattern of teratogenic effects, typical of exposure to triazoles, mainly consisting of reduction and fusion of the first and second branchial arches, which are in accordance with the craniofacial malformations reported after in vivo exposure. In the EST all triazole compounds inhibited cardiomyocyte differentiation concentration-dependently. Overall, the ZET gave the best correlation with the relative in vivo developmental toxicities of the tested compounds, closely followed by the EST. The relative potencies observed in the WEC showed the lowest correlation with the in vivo developmental toxicity data. These differences in the efficacy between the test systems might be due to differences in compound kinetics, in developmental stages represented and in the relative complexity of the alternative assays.

Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes

Energy Technology Data Exchange (ETDEWEB)

Marion, Tracy L., E-mail: tracylmarion@qualyst.com [Curriculum in Toxicology, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7270 (United States); Perry, Cassandra H., E-mail: cassandraperry@qualyst.com [Qualyst, Inc., Durham, NC 27713 (United States); St Claire, Robert L., E-mail: bobstclaire@qualyst.com [Qualyst, Inc., Durham, NC 27713 (United States); Brouwer, Kim L.R., E-mail: kbrouwer@unc.edu [Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, CB 7569 Kerr Hall, Chapel Hill, NC 27599-7569 (United States)

2012-05-15

Sandwich-cultured hepatocytes (SCH) are used commonly to investigate hepatic transport protein-mediated uptake and biliary excretion of substrates. However, little is known about the disposition of endogenous bile acids (BAs) in SCH. In this study, four endogenous conjugated BAs common to rats and humans [taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA)], as well as two BA species specific to rodents (α- and β-tauromuricholic acid; α/β TMCA), were profiled in primary rat and human SCH. Using B-CLEAR{sup ®} technology, BAs were measured in cells + bile canaliculi, cells, and medium of SCH by LC-MS/MS. Results indicated that, just as in vivo, taurine-conjugated BA species were predominant in rat SCH, while glycine-conjugated BAs were predominant in human SCH. Total intracellular BAs remained relatively constant over days in culture in rat SCH. Total BAs in control (CTL) cells + bile, cells, and medium were approximately 3.4, 2.9, and 8.3-fold greater in human than in rat. The estimated intracellular concentrations of the measured total BAs were 64.3 ± 5.9 μM in CTL rat and 183 ± 56 μM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16 ± 0.21 μM in CTL rat SCH and 9.61 ± 6.36 μM in CTL human SCH. Treatment of cells for 24 h with 10 μM troglitazone (TRO), an inhibitor of the bile salt export pump (BSEP) and the Na{sup +}-taurocholate cotransporting polypeptide (NTCP), had no significant effect on endogenous BAs measured at the end of the 24-h culture period, potentially due to compensatory mechanisms that maintain BA homeostasis. These data demonstrate that BAs in SCH are similar to in vivo, and that SCH may be a useful in vitro model to study alterations in BA disposition if species differences are taken into account. -- Highlights: ► Bile acids (BAs) were measured in rat and human sandwich-cultured hepatocytes (SCH). ► Cell and medium BA

Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes

International Nuclear Information System (INIS)

Marion, Tracy L.; Perry, Cassandra H.; St Claire, Robert L.; Brouwer, Kim L.R.

2012-01-01

Sandwich-cultured hepatocytes (SCH) are used commonly to investigate hepatic transport protein-mediated uptake and biliary excretion of substrates. However, little is known about the disposition of endogenous bile acids (BAs) in SCH. In this study, four endogenous conjugated BAs common to rats and humans [taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA)], as well as two BA species specific to rodents (α- and β-tauromuricholic acid; α/β TMCA), were profiled in primary rat and human SCH. Using B-CLEAR ® technology, BAs were measured in cells + bile canaliculi, cells, and medium of SCH by LC-MS/MS. Results indicated that, just as in vivo, taurine-conjugated BA species were predominant in rat SCH, while glycine-conjugated BAs were predominant in human SCH. Total intracellular BAs remained relatively constant over days in culture in rat SCH. Total BAs in control (CTL) cells + bile, cells, and medium were approximately 3.4, 2.9, and 8.3-fold greater in human than in rat. The estimated intracellular concentrations of the measured total BAs were 64.3 ± 5.9 μM in CTL rat and 183 ± 56 μM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16 ± 0.21 μM in CTL rat SCH and 9.61 ± 6.36 μM in CTL human SCH. Treatment of cells for 24 h with 10 μM troglitazone (TRO), an inhibitor of the bile salt export pump (BSEP) and the Na + -taurocholate cotransporting polypeptide (NTCP), had no significant effect on endogenous BAs measured at the end of the 24-h culture period, potentially due to compensatory mechanisms that maintain BA homeostasis. These data demonstrate that BAs in SCH are similar to in vivo, and that SCH may be a useful in vitro model to study alterations in BA disposition if species differences are taken into account. -- Highlights: ► Bile acids (BAs) were measured in rat and human sandwich-cultured hepatocytes (SCH). ► Cell and medium BA concentrations

Dose-response analysis of phthalate effects on gene expression in rat whole embryo culture

NARCIS (Netherlands)

Robinson, J.F.; Verhoef, A.; van Beelen, V.A.; Pennings, J.L.A.; Piersma, A.H.|info:eu-repo/dai/nl/071276947

2012-01-01

The rat postimplantation whole embryo culture (WEC) model serves as a potential screening tool for developmental toxicity. In this model, cultured rat embryos are exposed during early embryogenesis and evaluated for morphological effects. The integration of molecular-based markers may lead to

Identification of rat Rosa26 locus enables generation of knock-in rat lines ubiquitously expressing tdTomato.

Science.gov (United States)

Kobayashi, Toshihiro; Kato-Itoh, Megumi; Yamaguchi, Tomoyuki; Tamura, Chihiro; Sanbo, Makoto; Hirabayashi, Masumi; Nakauchi, Hiromitsu

2012-11-01

Recent discovery of a method for derivation and culture of germline-competent rat pluripotent stem cells (PSCs) enables generation of transgenic rats or knock-out rats via genetic modification of such PSCs. This opens the way to use rats, as is routine in mice, for analyses of gene functions or physiological features. In mouse or human, one widely used technique to express a gene of interest stably and ubiquitously is to insert that gene into the Rosa26 locus via gene targeting of PSCs. Rosa26 knock-in mice conditionally expressing a reporter or a toxin gene have contributed to tracing or ablation of specific cell lineages. We successfully identified a rat orthologue of the mouse Rosa26 locus. Insertion of tdTomato, a variant of red fluorescent protein, into the Rosa26 locus of PSCs of various rat strains allows ubiquitous expression of tdTomato. Through germline transmission of one Rosa26-tdTomato knock-in embryonic stem cell line, we also obtained tdTomato knock-in rats. These expressed tdTomato ubiquitously throughout their bodies, which indicates that the rat Rosa26 locus conserves functions of its orthologues in mouse and human. The new tools described here (targeting vectors, knock-in PSCs, and rats) should be useful for a variety of research using rats.

Rat brain sagittal organotypic slice cultures as an ex vivo dopamine cell loss system.

Science.gov (United States)

McCaughey-Chapman, Amy; Connor, Bronwen

2017-02-01

Organotypic brain slice cultures are a useful tool to study neurological function as they provide a more complex, 3-dimensional system than standard 2-dimensional in vitro cell cultures. Building on a previously developed mouse brain slice culture protocol, we have developed a rat sagittal brain slice culture system as an ex vivo model of dopamine cell loss. We show that rat brain organotypic slice cultures remain viable for up to 6 weeks in culture. Using Fluoro-Gold axonal tracing, we demonstrate that the slice 3-dimensional cytoarchitecture is maintained over a 4 week culturing period, with particular focus on the nigrostriatal pathway. Treatment of the cultures with 6-hydroxydopamine and desipramine induces a progressive loss of Fluoro-Gold-positive nigral cells with a sustained loss of tyrosine hydroxylase-positive nigral cells. This recapitulates the pattern of dopaminergic degeneration observed in the rat partial 6-hydroxydopamine lesion model and, most importantly, the progressive pathology of Parkinson's disease. Our slice culture platform provides an advance over other systems, as we demonstrate for the first time 3-dimensional cytoarchitecture maintenance of rat nigrostriatal sagittal slices for up to 6 weeks. Our ex vivo organotypic slice culture system provides a long term cellular platform to model Parkinson's disease, allowing for the elucidation of mechanisms involved in dopaminergic neuron degeneration and the capability to study cellular integration and plasticity ex vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of p75{sup +} ectomesenchymal stem cells from rat embryonic facial process tissue

Energy Technology Data Exchange (ETDEWEB)

Wen, Xiujie; Liu, Luchuan; Deng, Manjing; Zhang, Li; Liu, Rui; Xing, Yongjun; Zhou, Xia [Department of Stomatology, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing 400042 (China); Nie, Xin, E-mail: dr.xinnie@gmail.com [Department of Stomatology, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing 400042 (China)

2012-10-12

Highlights: Black-Right-Pointing-Pointer Ectomesenchymal stem cells (EMSCs) were found to migrate to rat facial processes at E11.5. Black-Right-Pointing-Pointer We successfully sorted p75NTR positive EMSCs (p75{sup +} EMSCs). Black-Right-Pointing-Pointer p75{sup +} EMSCs up to nine passages showed relative stable proliferative activity. Black-Right-Pointing-Pointer We examined the in vitro multilineage potential of p75{sup +} EMSCs. Black-Right-Pointing-Pointer p75{sup +}EMSCs provide an in vitro model for tooth morphogenesis. -- Abstract: Several populations of stem cells, including those from the dental pulp and periodontal ligament, have been isolated from different parts of the tooth and periodontium. The characteristics of such stem cells have been reported as well. However, as a common progenitor of these cells, ectomesenchymal stem cells (EMSCs), derived from the cranial neural crest have yet to be fully characterized. The aim of this study was to better understand the characteristics of EMSCs isolated from rat embryonic facial processes. Immunohistochemical staining showed that EMSCs had migrated to rat facial processes at E11.5, while the absence of epithelial invagination or tooth-like epithelium suggested that any epithelial-mesenchymal interactions were limited at this stage. The p75 neurotrophin receptor (p75NTR), a typical neural crest marker, was used to select p75NTR-positive EMSCs (p75{sup +} EMSCs), which were found to show a homogeneous fibroblast-like morphology and little change in the growth curve, proliferation capacity, and cell phenotype during cell passage. They also displayed the capacity to differentiate into diverse cell types under chemically defined conditions in vitro. p75{sup +} EMSCs proved to be homogeneous, stable in vitro and potentially capable of multiple lineages, suggesting their potential for application in dental or orofacial tissue engineering.

Inhibition of chondroitin sulfate glycosaminoglycans incorporation affected odontoblast differentiation in cultured embryonic mouse molars.

Science.gov (United States)

Liu, Lipei; Chen, Weiting; Li, Lefeng; Xu, Fangfang; Jiang, Beizhan

2017-12-01

Chondroitin sulfate proteoglycan (CSPG) is an important component of extracellular matrix (ECM), it is composed of a core protein and one or more chondroitin sulfate glycosaminoglycan side chains (CS-GAGs). To investigate the roles of its CS-GAGs in dentinogenesis, the mouse mandibular first molar tooth germs at early bell stage were cultivated with or without β-xyloside. As expected, the CS-GAGs were inhibited on their incorporation to CSPGs by β-xyloside, accompanied by the change of morphology of the cultured tooth germs. The histological results and the transmission electron microscopy (TEM) investigation indicated that β-xyloside exhibited obvious inhibiting effects on odontoblasts differentiation compared with the control group. Meanwhile the results of immunohistochemistry, in situ hybridization and quantitative RT-PCR for type I collagen, dentin matrix acidic phosphoprotein 1 and dentin sialophosphoprotein, the products of differentiated odontoblasts, further proved that odontoblasts differentiation was inhibited. Collagen fibers detected in TEM decreased and arranged in disorder as well. Thus we conclude that the inhibition of CS-GAGs incorporation to CSPGs can affect odontoblast differentiation in cultured embryonic mouse molars.

Upregulation of endothelin ETB receptor-mediated vasoconstriction in rat coronary artery after organ culture

DEFF Research Database (Denmark)

Eskesen, Karen; Edvinsson, Lars

2006-01-01

The aim of this study was to examine if endothelin ET(B) receptor-mediated contraction occurred in isolated segments of rat coronary arteries during organ culture. Presence of contractile endothelin ET(B) receptors was studied by measuring the change in isometric tension in rings of left anterior......(+)-solution was not modified after 1 day in culture medium. The experiments indicate that organ culture of rat coronary arteries upregulate endothelin ET(B) receptor-mediated contraction by inducing synthesis of new protein....... descending coronary arteries isolated from hearts of rats as response to application of the selective endothelin ET(B) receptor agonist, Sarafotoxin 6c and endothelin-1. In segments cultured 1 day in serum free Dulbecco's Modified Eagle's Medium, Sarafotoxin 6c induced a concentration dependent contraction...

PKC/CREB pathway mediates the expressions of GABAA receptor subunits in cultured hippocampal neurons after low-Mg2+ solution treatment.

Science.gov (United States)

Wu, Guofeng; Yu, Jinpeng; Wang, Likun; Ren, Siying; Zhang, Yixia

2018-02-01

To investigate the potential effects of the PKC/CREB pathway on the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons using a model of epilepsy that employed conditions of low magnesium (Mg 2+ ). A total of 108 embryonic rats at the age of 18 embryonic days (E18)prepared from adult female SD rats were used as experimental subjects. Primary rat hippocampal cultures were prepared from the embryonic 18 days rats. The cultured hippocampal neurons were then treated with artificial cerebrospinal fluid containing low Mg 2+ solutions to generate a low Mg 2+ model of epilepsy. The low Mg 2+ stimulation lasted for 3 h and then returned to in maintenance medium for 20 h. The changes of the GABA A receptor subunit α1, γ2, δ were observed by blocking or activating the function of the CREB. The quantification of the GABA A receptor subunit α1, γ2, δ and the CREB were determined by a qRT-PCR and a Western blot method. After the neurons were exposed to a low-Mg 2+ solution for 3 h, GABA A receptor mRNA expression markedly increased compared to the control, and then gradually decreased. In contrast, CREB mRNA levels exhibited a dramatic down-regulation 3 h after terminating low-Mg 2+ treatment, and then peaked at 9 h. Western blot analyses verified that staurosporine suppressed CREB phosphorylation (p-CREB). The mRNA expression of GABA A receptor subunit α1 increased only in the presence of staurosporine, whereas the expressions of subunits γ2 and δ significantly increased in the presence of either KG-501 or staurosporine. Furthermore, phorbol 12-myristate 13-acetate (PMA) decreased the expressions of GABA A subunits α1, γ2, and δ when administered alone. However, the administration of either KG-501 or staurosporine reversed the inhibitory effects of PMA. The PKC/CREB pathway may negatively regulate the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons in low Mg 2+ model of

A comparative study of myosin and its subunits in adult and neonatal-rat hearts and in rat heart cells from young and old cultures.

OpenAIRE

Ghanbari, H A; McCarl, R L

1980-01-01

A possible explanation for the decrease in myosin Ca2+-dependent ATPase activity as rat heart cells age in culture is presented. The subunit structure and enzyme kinetics of myosin from adult and neonatal rat hearts and from rat heart cells of young and old cultures are compared. These studies indicate that the loss in Ca-ATPase activity of myosin from older cultures was an intrinsic property of the myosin itself. Myofibrillar fractions from the indicated four sources showed no qualitative or...

Measurement of contractile stress generated by cultured rat muscle on silicon cantilevers for toxin detection and muscle performance enhancement.

Directory of Open Access Journals (Sweden)

Kerry Wilson

2010-06-01

Full Text Available To date, biological components have been incorporated into MEMS devices to create cell-based sensors and assays, motors and actuators, and pumps. Bio-MEMS technologies present a unique opportunity to study fundamental biological processes at a level unrealized with previous methods. The capability to miniaturize analytical systems enables researchers to perform multiple experiments in parallel and with a high degree of control over experimental variables for high-content screening applications.We have demonstrated a biological microelectromechanical system (BioMEMS based on silicon cantilevers and an AFM detection system for studying the physiology and kinetics of myotubes derived from embryonic rat skeletal muscle. It was shown that it is possible to interrogate and observe muscle behavior in real time, as well as selectively stimulate the contraction of myotubes with the device. Stress generation of the tissue was estimated using a modification of Stoney's equation. Calculated stress values were in excellent agreement with previously published results for cultured myotubes, but not adult skeletal muscle. Other parameters such as time to peak tension (TPT, the time to half relaxation ((1/2RT were compared to the literature. It was observed that the myotubes grown on the BioMEMS device, while generating stress magnitudes comparable to those previously published, exhibited slower TPT and (1/2RT values. However, growth in an enhanced media increased these values. From these data it was concluded that the myotubes cultured on the cantilevers were of an embryonic phenotype. The system was also shown to be responsive to the application of a toxin, veratridine.The device demonstrated here will provide a useful foundation for studying various aspects of muscle physiology and behavior in a controlled high-throughput manner as well as be useful for biosensor and drug discovery applications.

In situ hybridization analysis of the temporospatial expression of the midkine/pleiotrophin family in rat embryonic pituitary gland.

Science.gov (United States)

Fujiwara, Ken; Maliza, Rita; Tofrizal, Alimuddin; Batchuluun, Khongorzul; Ramadhani, Dini; Tsukada, Takehiro; Azuma, Morio; Horiguchi, Kotaro; Kikuchi, Motoshi; Yashiro, Takashi

2014-07-01

Pituitary gland development is controlled by numerous signaling molecules, which are produced in the oral ectoderm and diencephalon. A newly described family of heparin-binding growth factors, namely midkine (MK)/pleiotrophin (PTN), is involved in regulating the growth and differentiation of many tissues and organs. Using in situ hybridization with digoxigenin-labeled cRNA probes, we detected cells expressing MK and PTN in the developing rat pituitary gland. At embryonic day 12.5 (E12.5), MK expression was localized in Rathke's pouch (derived from the oral ectoderm) and in the neurohypophyseal bud (derived from the diencephalon). From E12.5 to E19.5, MK mRNA was expressed in the developing neurohypophysis, and expression gradually decreased in the developing adenohypophysis. To characterize MK-expressing cells, we performed double-staining of MK mRNA and anterior pituitary hormones. At E19.5, no MK-expressing cells were stained with any hormone. In contrast, PTN was expressed only in the neurohypophysis primordium during all embryonic stages. In situ hybridization clearly showed that MK was expressed in primitive (immature/undifferentiated) adenohypophyseal cells and neurohypophyseal cells, whereas PTN was expressed only in neurohypophyseal cells. Thus, MK and PTN might play roles as signaling molecules during pituitary development.

Hepatic esterase activity is increased in hepatocyte-like cells derived from human embryonic stem cells using a 3D culture system.

Science.gov (United States)

Choi, Young-Jun; Kim, Hyemin; Kim, Ji-Woo; Yoon, Seokjoo; Park, Han-Jin

2018-05-01

The aim of the study is to generate a spherical three-dimensional (3D) aggregate of hepatocyte-like cells (HLCs) differentiated from human embryonic stem cells and to investigate the effect of the 3D environment on hepatic maturation and drug metabolism. Quantitative real-time PCR analysis indicated that gene expression of mature hepatocyte markers, drug-metabolizing enzymes, and hepatic transporters was significantly higher in HLCs cultured in the 3D system than in those cultured in a two-dimensional system (p formation, were increased in HLCs cultured in the 3D system. In particular, 3D spheroidal culture increased expression of CES1 and BCHE, which encode hepatic esterases (p 3D spheroidal culture enhances the maturation and drug metabolism of stem cell-derived HLCs, and this may help to optimize hepatic differentiation protocols for hepatotoxicity testing.

Impaired embryonic development in mice overexpressing the RNA-binding protein TIAR.

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Yacine Kharraz

Full Text Available BACKGROUND: TIA-1-related (TIAR protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs. Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translational arrest observed in cells exposed to environmental stress. However, the in vivo role of this protein has not been studied so far mainly due to severe embryonic lethality upon tiar invalidation. METHODOLOGY/PRINCIPAL FINDINGS: To examine potential TIAR tissue-specificity in various cellular contexts, either embryonic or adult, we constructed a TIAR transgenic allele (loxPGFPloxPTIAR allowing the conditional expression of TIAR protein upon Cre recombinase activity. Here, we report the role of TIAR during mouse embryogenesis. We observed that early TIAR overexpression led to low transgene transmission associated with embryonic lethality starting at early post-implantation stages. Interestingly, while pre-implantation steps evolved correctly in utero, in vitro cultured embryos were very sensitive to culture medium. Control and transgenic embryos developed equally well in the G2 medium, whereas culture in M16 medium led to the phosphorylation of eIF2alpha that accumulated in cytoplasmic granules precluding transgenic blastocyst hatching. Our results thus reveal a differential TIAR-mediated embryonic response following artificial or natural growth environment. CONCLUSIONS/SIGNIFICANCE: This study reports the importance of the tightly balanced expression of the RNA-binding protein TIAR for normal embryonic development, thereby emphasizing the role of post-transcriptional regulations in early embryonic programming.

Cyanidin-3-glucoside inhibits glutamate-induced Zn2+ signaling and neuronal cell death in cultured rat hippocampal neurons by inhibiting Ca2+-induced mitochondrial depolarization and formation of reactive oxygen species.

Science.gov (United States)

Yang, Ji Seon; Perveen, Shazia; Ha, Tae Joung; Kim, Seong Yun; Yoon, Shin Hee

2015-05-05

Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is a potent natural antioxidant. However, effects of C3G on glutamate-induced [Zn(2+)]i increase and neuronal cell death remain unknown. We studied the effects of C3G on glutamate-induced [Zn(2+)]i increase and cell death in cultured rat hippocampal neurons from embryonic day 17 maternal Sprague-Dawley rats using digital imaging methods for Zn(2+), Ca(2+), reactive oxygen species (ROS), mitochondrial membrane potential and a MTT assay for cell survival. Treatment with glutamate (100 µM) for 7 min induces reproducible [Zn(2+)]i increase at 35 min interval in cultured rat hippocampal neurons. The intracellular Zn(2+)-chelator TPEN markedly blocked glutamate-induced [Zn(2+)]i increase, but the extracellular Zn(2+) chelator CaEDTA did not affect glutamate-induced [Zn(2+)]i increase. C3G inhibited the glutamate-induced [Zn(2+)]i response in a concentration-dependent manner (IC50 of 14.1 ± 1.1 µg/ml). C3G also significantly inhibited glutamate-induced [Ca(2+)]i increase. Two antioxidants such as Trolox and DTT significantly inhibited the glutamate-induced [Zn(2+)]i response, but they did not affect the [Ca(2+)]i responses. C3G blocked glutamate-induced formation of ROS. Trolox and DTT also inhibited the formation of ROS. C3G significantly inhibited glutamate-induced mitochondrial depolarization. However, TPEN, Trolox and DTT did not affect the mitochondrial depolarization. C3G, Trolox and DTT attenuated glutamate-induced neuronal cell death in cultured rat hippocampal neurons, respectively. Taken together, all these results suggest that cyanidin-3-glucoside inhibits glutamate-induced [Zn(2+)]i increase through a release of Zn(2+) from intracellular sources in cultured rat hippocampal neurons by inhibiting Ca(2+)-induced mitochondrial depolarization and formation of ROS, which is involved in neuroprotection against glutamate-induced cell death. Copyright © 2015 Elsevier B.V. All rights reserved.

Primary Bovine Extra-Embryonic Cultured Cells: A New Resource for the Study of In Vivo Peri-Implanting Phenotypes and Mesoderm Formation.

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Isabelle Hue

Full Text Available In addition to nourishing the embryo, extra-embryonic tissues (EETs contribute to early embryonic patterning, primitive hematopoiesis, and fetal health. These tissues are of major importance for human medicine, as well as for efforts to improve livestock efficiency, but they remain incompletely understood. In bovines, EETs are accessible easily, in large amounts, and prior to implantation. We took advantage of this system to describe, in vitro and in vivo, the cell types present in bovine EETs at Day 18 of development. Specifically, we characterized the gene expression patterns and phenotypes of bovine extra-embryonic ectoderm (or trophoblast; bTC, endoderm (bXEC, and mesoderm (bXMC cells in culture and compared them to their respective in vivo micro-dissected cells. After a week of culture, certain characteristics (e.g., gene expression of the in vitro cells were altered with respect to the in vivo cells, but we were able to identify "cores" of cell-type-specific (and substrate-independent genes that were shared between in vitro and in vivo samples. In addition, many cellular phenotypes were cell-type-specific with regard to extracellular adhesion. We evaluated the ability of individual bXMCs to migrate and spread on micro-patterns, and observed that they easily adapted to diverse environments, similar to in vivo EE mesoderm cells, which encounter different EE epithelia to form chorion, yolk sac, and allantois. With these tissue interactions, different functions arose that were detected in silico and corroborated in vivo at D21-D25. Moreover, analysis of bXMCs allowed us to identify the EE cell ring surrounding the embryonic disc (ED at D14-15 as mesoderm cells, which had been hypothesized but not shown prior to this study. We envision these data will serve as a major resource for the future in the analysis of peri-implanting phenotypes in response to the maternal metabolism and contribute to subsequent studies of placental/fetal development in

Chromosome preparations from microplate cultures of man, dog, rat, and mouse

NARCIS (Netherlands)

de Jong, B; Anders, GJPA; van der Meer, Ingrid H

1976-01-01

A simple method for making chromosomal preparations from 105 leukocytes from man, dog, mouse, and rat and from 0.01 ml total human and dog blood is developed. The leukocytes and the peripheral blood are cultured in Cooke microtiter plates in a culture volume of 0.1 ml. The culture medium is R.P.M.I.

Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells

DEFF Research Database (Denmark)

Prokhorova, Tatyana A; Rigbolt, Kristoffer T G; Johansen, Pia T

2009-01-01

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research...... embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers......: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase zeta (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell...

Differentiation of mouse embryonic stem cells into cardiomyocytes via the hanging-drop and mass culture methods.

Science.gov (United States)

Fuegemann, Christopher J; Samraj, Ajoy K; Walsh, Stuart; Fleischmann, Bernd K; Jovinge, Stefan; Breitbach, Martin

2010-12-01

Herein, we describe two protocols for the in vitro differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. mESCs are pluripotent and can be differentiated into cells of all three germ layers, including cardiomyocytes. The methods described here facilitate the differentiation of mESCs into the different cardiac subtypes (atrial-, ventricular-, nodal-like cells). The duration of cell culture determines whether preferentially early- or late-developmental stage cardiomyocytes can be obtained preferentially. This approach allows the investigation of cardiomyocyte development and differentiation in vitro, and also allows for the enrichment and isolation of physiologically intact cardiomyocytes for transplantation purposes. © 2010 by John Wiley & Sons, Inc.

Evaluation of hollow fiber culture for large-scale production of mouse embryonic stem cell-derived hematopoietic stem cells.

Science.gov (United States)

Nakano, Yu; Iwanaga, Shinya; Mizumoto, Hiroshi; Kajiwara, Toshihisa

2018-03-03

Hematopoietic stem cells (HSCs) have the ability to differentiate into all types of blood cells and can be transplanted to treat blood disorders. However, it is difficult to obtain HSCs in large quantities because of the shortage of donors. Recent efforts have focused on acquiring HSCs by differentiation of pluripotent stem cells. As a conventional differentiation method of pluripotent stem cells, the formation of embryoid bodies (EBs) is often employed. However, the size of EBs is limited by depletion of oxygen and nutrients, which prevents them from being efficient for the production of HSCs. In this study, we developed a large-scale hematopoietic differentiation approach for mouse embryonic stem (ES) cells by applying a hollow fiber (HF)/organoid culture method. Cylindrical organoids, which had the potential for further spontaneous differentiation, were established inside of hollow fibers. Using this method, we improved the proliferation rate of mouse ES cells to produce an increased HSC population and achieved around a 40-fold higher production volume of HSCs in HF culture than in conventional EB culture. Therefore, the HF/organoid culture method may be a new mass culture method to acquire pluripotent stem cell-derived HSCs.

In vitro differentiation of rat spermatogonia into round spermatids in tissue culture.

Science.gov (United States)

Reda, A; Hou, M; Winton, T R; Chapin, R E; Söder, O; Stukenborg, J-B

2016-09-01

Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in

Trimethyltin (TMT) neurotoxicity in organotypic rat hippocampal slice cultures

DEFF Research Database (Denmark)

Noraberg, J; Gramsbergen, J B; Fonnum, F

1998-01-01

) propidium iodide (PI) uptake, (b) lactate dehydrogenase (LDH) efflux into the culture medium, (c) cellular cobalt uptake as an index of calcium influx, (d) ordinary Nissl cell staining, and (e) immunohistochemical staining for microtubule-associated protein 2 (MAP-2). Cellular degeneration as assessed...... to in vivo cell stain observations of rats acutely exposed to TMT. The mean PI uptake of the cultures and the LDH efflux into the medium were highly correlated. The combined results obtained by the different markers indicate that the hippocampal slice culture method is a feasible model for further studies...

Inducibility of carbamoylphosphate synthetase (ammonia) in cultures of embryonic hepatocytes: ontogenesis of the responsiveness to hormones

NARCIS (Netherlands)

Lamers, W. H.; Zonneveld, D.; Charles, R.

1984-01-01

Glucocorticosteroids and cyclic AMP induce carbamoylphosphate synthetase (ammonia) (CPS) in rat hepatocytes. Using an enzyme immunoassay applied to hepatocyte cultures fixed in situ, it has been demonstrated that the capacity of hepatocytes to synthesize CPS in the presence of both hormones is

Differentiation of cartilaginous anlagen in entire embryonic mouse limbs cultured in a rotating bioreactor

Science.gov (United States)

Montufar-Solis, D.; Oakley, C. R.; Jefferson, Y.; Duke, P. J.

2003-10-01

Mechanisms involved in development of the embryonic limb have remained the same throughout eons of genetic and environmental evolution under Earth gravity (lg). During the spaceflight era it has been of interest to explore the ancient theory that form of the skeleton develops in response to gravity, and that changes in gravitational forces can change the developmental pattern of the limb. This has been shown in vivo and in vitro, allowing the hypergravity of centrifugation and microgravity of space to be used as tools to increase our knowledge of limb development. In recapitulations of spaceflight experiments, premetatarsals were cultured in suspension in a bioreactor, and found to be shorter and less differentiated than those cultured in standard culture dishes. This study only measured length of the metatarsals, and did not account for possible changes due to the skeletal elements having a more in vivo 3D shape while in suspension vs. flattened tissues compressed by their own weight. A culture system with an outcome closer to in vivo and that supports growth of younger limb buds than traditional systems will allow studies of early Hox gene expression, and contribute to the understanding of very early stages of development. The purpose of the current experiment was to determine if entire limb buds could be cultured in the bioreactor, and to compare the growth and differentiation with that of culturing in a culture dish system. Fore and hind limbs from E11-E13 ICR mouse embryos were cultured for six days, either in the bioreactor or in center-well organ culture dishes, fixed, and embedded for histology. E13 specimens grown in culture dishes were flat, while bioreactor culture specimens had a more in vivo-like 3D limb shape. Sections showed excellent cartilage differentiation in both culture systems, with more cell maturation, and hypertrophy in the specimens cultured in the bioreactor. Younger limb buds fused together during culture, so an additional set of El 1

A study to determine the relationship between the P-uptake and the number of embryonic roots by wheat CV Azadi

International Nuclear Information System (INIS)

Ahmadi, E.N.

1982-01-01

To find out the relationship between the number of the embryonic roots and the P-uptake and P-distribution a test was carried out with 18 days old wheat plants cv.''AZADI''in two culture solutions of different concentrations labeled with 32P (3,2μCi/1). After 48 hours uptake the 32P-measurement had shown, that the content of 32P was, by plants grown in both culture solutions, in roots higher than in shoots. The velocity of 32P uptake per root increased in both plant groups when the number of embryonic roots decreased, whereas by plants grown in culture solution with lower concentration, this increase could not compensate the P-shortage in the culture solution. Hence the quantitative 32P uptake per plant rose when the number of embryonic roots increased. Because of the importance of the number of embryonic roots under Iranian cultivation conditions it has been suggested, that the''number of embryonic roots'' should be considered as an important factor in wheat breeding programms. (Author)

Human embryonic stem cells and microenvironment

Directory of Open Access Journals (Sweden)

Banu İskender

2014-09-01

Full Text Available Human embryonic stem cells (hESCs possess a great potential in the field of regenerative medicine by their virtue of pluripotent potential with indefinite proliferation capabilities. They can self renew themselves and differentiate into three embryonic germ layers. Although they are conventionally grown on mitotically inactivated mouse feeder cells, there are in vitro culture systems utilizing feeder cells of human origin in order to prevent cross-species contamination. Recently established in vitro culture systems suggested that direct interaction with feeder cells is not necessary but rather attachment to a substrate is required to ensure long-term, efficient hESC culture in vitro. This substrate is usually composed of a mixture of extracellular matrix components representing in vivo natural niche. In hESC biology, the mechanism of interaction of hESCs with extracellular matrix molecules remained insufficiently explored area of research due to their transient nature of interaction with the in vivo niche. However, an in vitro culture system established using extracellular matrix molecules may provide a safer alternative to culture systems with feeder cells while paving the way to Good Manufacturing Practice-GMP production of hESCs for therapeutic purposes. Therefore, it is essential to study the interaction of extracellular matrix molecules with hESCs in order to standardize in vitro culture systems for large-scale production of hESCs in a less labor-intensive way. This would not only provide valuable information regarding the mechanisms that control pluripotency but also serve to dissect the molecular signaling pathways of directed differentiation for prospective therapeutic applications in the future. J Clin Exp Invest 2014; 5 (3: 486-495

Morphological and behavioral evaluation of central nervous system toxicity after embryonic x-irradiation

International Nuclear Information System (INIS)

Schneider, B.F.

1979-01-01

The purpose of these studies was to quantify the effects of embryonic x-irradiation on neuronal development and to describe the behavioral effects of such a treatment. Rats were exposed to 125r x-irradiation on gestational day 15

Avian influenza virus isolation, propagation and titration in embryonated chicken eggs

Science.gov (United States)

Avian influenza (AI) virus is usually isolated, propagated, and titrated in embryonated chickens eggs (ECE). Most any sample type can be accommodated for culture with appropriate processing. Isolation may also be accomplished in cell culture particularly if mammalian lineage isolates are suspected, ...

Improved genetic manipulation of human embryonic stem cells.

NARCIS (Netherlands)

Braam, S.R.; Denning, C.; van den Brink, S.; Kats, P.; Hochstenbach, R.; Passier, R.; Mummery, C.L.

2008-01-01

Low efficiency of transfection limits the ability to genetically manipulate human embryonic stem cells (hESCs), and differences in cell derivation and culture methods require optimization of transfection protocols. We transiently transferred multiple independent hESC lines with different growth

Comparison of neurotrophin and repellent sensitivities of early embryonic geniculate and trigeminal axons

NARCIS (Netherlands)

Rochlin, M William; O'Connor, R.; Giger, Roman J; Verhaagen, J; Farbman, A I

2000-01-01

Geniculate (gustatory) and trigeminal (somatosensory) afferents take different routes to the tongue during rat embryonic development. To learn more about the mechanisms controlling neurite outgrowth and axon guidance, we are studying the roles of diffusible factors. We previously profiled the in

Stepwise development of hematopoietic stem cells from embryonic stem cells.

Directory of Open Access Journals (Sweden)

Kenji Matsumoto

Full Text Available The cellular ontogeny of hematopoietic stem cells (HSCs remains poorly understood because their isolation from and their identification in early developing small embryos are difficult. We attempted to dissect early developmental stages of HSCs using an in vitro mouse embryonic stem cell (ESC differentiation system combined with inducible HOXB4 expression. Here we report the identification of pre-HSCs and an embryonic type of HSCs (embryonic HSCs as intermediate cells between ESCs and HSCs. Both pre-HSCs and embryonic HSCs were isolated by their c-Kit(+CD41(+CD45(- phenotype. Pre-HSCs did not engraft in irradiated adult mice. After co-culture with OP9 stromal cells and conditional expression of HOXB4, pre-HSCs gave rise to embryonic HSCs capable of engraftment and long-term reconstitution in irradiated adult mice. Blast colony assays revealed that most hemangioblast activity was detected apart from the pre-HSC population, implying the early divergence of pre-HSCs from hemangioblasts. Gene expression profiling suggests that a particular set of transcripts closely associated with adult HSCs is involved in the transition of pre-HSC to embryonic HSCs. We propose an HSC developmental model in which pre-HSCs and embryonic HSCs sequentially give rise to adult types of HSCs in a stepwise manner.

Diabetes increases susceptibility of primary cultures of rat proximal tubular cells to chemically induced injury

International Nuclear Information System (INIS)

Zhong Qing; Terlecky, Stanley R.; Lash, Lawrence H.

2009-01-01

Diabetic nephropathy is characterized by increased oxidative stress and mitochondrial dysfunction. In the present study, we prepared primary cultures of proximal tubular (PT) cells from diabetic rats 30 days after an ip injection of streptozotocin and compared their susceptibility to oxidants (tert-butyl hydroperoxide, methyl vinyl ketone) and a mitochondrial toxicant (antimycin A) with that of PT cells isolated from age-matched control rats, to test the hypothesis that PT cells from diabetic rats exhibit more cellular and mitochondrial injury than those from control rats when exposed to these toxicants. PT cells from diabetic rats exhibited higher basal levels of reactive oxygen species (ROS) and higher mitochondrial membrane potential, demonstrating that the PT cells maintain the diabetic phenotype in primary culture. Incubation with either the oxidants or mitochondrial toxicant resulted in greater necrotic and apoptotic cell death, greater evidence of morphological damage, greater increases in ROS, and greater decreases in mitochondrial membrane potential in PT cells from diabetic rats than in those from control rats. Pretreatment with either the antioxidant N-acetyl-L-cysteine or a catalase mimetic provided equivalent protection of PT cells from both diabetic and control rats. Despite the greater susceptibility to oxidative and mitochondrial injury, both cytoplasmic and mitochondrial glutathione concentrations were markedly higher in PT cells from diabetic rats, suggesting an upregulation of antioxidant processes in diabetic kidney. These results support the hypothesis that primary cultures of PT cells from diabetic rats are a valid model in which to study renal cellular function in the diabetic state.

In vitro pancreas organogenesis from dispersed mouse embryonic progenitors

DEFF Research Database (Denmark)

Greggio, Chiara; De Franceschi, Filippo; Figueiredo-Larsen, Evan Manuel

2014-01-01

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells (1). The whole embryonic organ can be cultured at multiple stages...... expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how...... cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess...

Study on the effect of hypoxia on apoptosis of cultured newly born rat cardiac myocytes

International Nuclear Information System (INIS)

Su Weidong; Li Huiqiang; Yao Zhi

2005-01-01

Objective: To investigate the possible hypoxia-mediated cellular apoptosis after ischemic cardiac injury via a model of cultured newly born rat cardiac myocytes. Methods: Cardiac myocytes cultures from newly born rats (1-3d) were examined for apoptosis with HE stain and flow cytometry after cultured 96h and again examined after exposure to hypoxic environment for 16h. Results: Apoptotic changes were evident in the hypoxic culture cells. The HE stain revealed cellular shrinkage, nuclear chromosomal condensation with cytoplasmic eosinophilia. Also, distinct apoptosis peak was observed in the flow cytometry. Conclusion: This experiment proved that hypoxic model of cardiac myocyte culture showed definite apoptosis of the cells. (authors)

Radiosensitivity of a epithelial cell model from an embryonic rat lung involving in particular the status of p53 gene

International Nuclear Information System (INIS)

Paris, Francois

1998-01-01

In this research thesis, the author presents ionizing radiations and their effects on living matter (damages to DNA, cell response to irradiation, proteins activated by radio-induced DNA breaks), the p53 protein (p53 mutation in cancers, structure), and the effect of ionizing radiation on this protein (expression and activation). Then this thesis addresses the study of a set of sister line of epithelial cells obtained from an embryonic rat lung treated with benzo(a)pyrene, a mutagenic agent notably present in cigarette smoke, in hydrocarbon combustion and in atmospheric pollution, and therefore responsible of cancers. This thesis thus reports the development of an experimental model allowing transformed cells to be studied [fr

Nitric oxide synthase-3 promotes embryonic development of atrioventricular valves.

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Yin Liu

Full Text Available Nitric oxide synthase-3 (NOS3 has recently been shown to promote endothelial-to-mesenchymal transition (EndMT in the developing atrioventricular (AV canal. The present study was aimed to investigate the role of NOS3 in embryonic development of AV valves. We hypothesized that NOS3 promotes embryonic development of AV valves via EndMT. To test this hypothesis, morphological and functional analysis of AV valves were performed in wild-type (WT and NOS3(-/- mice at postnatal day 0. Our data show that the overall size and length of mitral and tricuspid valves were decreased in NOS3(-/- compared with WT mice. Echocardiographic assessment showed significant regurgitation of mitral and tricuspid valves during systole in NOS3(-/- mice. These phenotypes were all rescued by cardiac specific NOS3 overexpression. To assess EndMT, immunostaining of Snail1 was performed in the embryonic heart. Both total mesenchymal and Snail1(+ cells in the AV cushion were decreased in NOS3(-/- compared with WT mice at E10.5 and E12.5, which was completely restored by cardiac specific NOS3 overexpression. In cultured embryonic hearts, NOS3 promoted transforming growth factor (TGFβ, bone morphogenetic protein (BMP2 and Snail1expression through cGMP. Furthermore, mesenchymal cell formation and migration from cultured AV cushion explants were decreased in the NOS3(-/- compared with WT mice. We conclude that NOS3 promotes AV valve formation during embryonic heart development and deficiency in NOS3 results in AV valve insufficiency.

Uptake of thyroxine in cultured anterior pituitary cells of euthyroid rats

NARCIS (Netherlands)

M.E. Everts (Maria); R. Docter (Roel); E.P.C.M. Moerings (Ellis); P.M. van Koetsveld (Peter); T.J. Visser (Theo); E.P. Krenning (Eric); G. Hennemann; M. de Jong (Marcel)

1994-01-01

textabstractThe uptake of [125I]T4 was investigated in cultured anterior pituitary cells isolated from adult fed Wistar rats and cultured for 3 days in medium containing 10% fetal calf serum. Experiments were performed with [125I]T4 (10(5) to 2 x 10(6) cpm; 0.35-7 nM)

The Therapeutic Effect of Human Embryonic Stem Cell-Derived Multipotent Mesenchymal Stem Cells on Chemical-Induced Cystitis in Rats

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Sang Wook Lee

2018-01-01

Full Text Available Purpose To evaluate the therapeutic effect of human embryonic stem cell (hESC-derived multipotent mesenchymal stem cells (M-MSCs on ketamine-induced cystitis (KC in rats. Methods To induce KC, 10-week-old female rats were injected with 25-mg/kg ketamine hydrochloride twice weekly for 12 weeks. In the sham group, phosphate buffered saline (PBS was injected instead of ketamine. One week after the final injection of ketamine, the indicated doses (0.25, 0.5, and 1×106 cells of M-MSCs (KC+M-MSC group or PBS vehicle (KC group were directly injected into the bladder wall. One week after M-MSC injection, the therapeutic outcomes were evaluated via cystometry, histological analyses, and measurement of gene expression. Next, we compared the efficacy of M-MSCs at a low dose (1×105 cells to that of an identical dose of adult bone marrow (BM-derived MSCs. Results Rats in the KC group exhibited increased voiding frequency and reduced bladder capacity compared to rats of the sham group. However, these parameters recovered after transplantation of M-MSCs at all doses tested. KC bladders exhibited markedly increased mast cell infiltration, apoptosis, and tissue fibrosis. Administration of M-MSCs significantly reversed these characteristic histological alterations. Gene expression analyses indicated that several genes associated with tissue fibrosis were markedly upregulated in KC bladders. However the expression of these genes was significantly suppressed by the administration of M-MSCs. Importantly, M-MSCs ameliorated bladder deterioration in KC rats after injection of a low dose (1×105 of cells, at which point BM-derived MSCs did not substantially improve bladder function. Conclusions This study demonstrates for the first time the therapeutic efficacy of hESC-derived M-MSCs on KC in rats. M-MSCs restored bladder function more effectively than did BM-derived MSCs, protecting against abnormal changes including mast cell infiltration, apoptosis and fibrotic

Teratoma formation of human embryonic stem cells in three-dimensional perfusion culture bioreactors.

Science.gov (United States)

Stachelscheid, H; Wulf-Goldenberg, A; Eckert, K; Jensen, J; Edsbagge, J; Björquist, P; Rivero, M; Strehl, R; Jozefczuk, J; Prigione, A; Adjaye, J; Urbaniak, T; Bussmann, P; Zeilinger, K; Gerlach, J C

2013-09-01

Teratoma formation in mice is today the most stringent test for pluripotency that is available for human pluripotent cells, as chimera formation and tetraploid complementation cannot be performed with human cells. The teratoma assay could also be applied for assessing the safety of human pluripotent cell-derived cell populations intended for therapeutic applications. In our study we examined the spontaneous differentiation behaviour of human embryonic stem cells (hESCs) in a perfused 3D multi-compartment bioreactor system and compared it with differentiation of hESCs and human induced pluripotent cells (hiPSCs) cultured in vitro as embryoid bodies and in vivo in an experimental mouse model of teratoma formation. Results from biochemical, histological/immunohistological and ultrastuctural analyses revealed that hESCs cultured in bioreactors formed tissue-like structures containing derivatives of all three germ layers. Comparison with embryoid bodies and the teratomas revealed a high degree of similarity of the tissues formed in the bioreactor to these in the teratomas at the histological as well as transcriptional level, as detected by comparative whole-genome RNA expression profiling. The 3D culture system represents a novel in vitro model that permits stable long-term cultivation, spontaneous multi-lineage differentiation and tissue formation of pluripotent cells that is comparable to in vivo differentiation. Such a model is of interest, e.g. for the development of novel cell differentiation strategies. In addition, the 3D in vitro model could be used for teratoma studies and pluripotency assays in a fully defined, controlled environment, alternatively to in vivo mouse models. Copyright © 2012 John Wiley & Sons, Ltd.

Three-Dimensional Culture of Human Embryonic Stem Cell Derived Hepatic Endoderm and Its Role in Bioartificial Liver Construction

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Ruchi Sharma

2010-01-01

Full Text Available The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays.

Self-contained induction of neurons from human embryonic stem cells.

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Tsuyoshi Okuno

Full Text Available BACKGROUND: Neurons and glial cells can be efficiently induced from mouse embryonic stem (ES cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM. However, the use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an alternative method for unimpeded production of human neurons under xeno-free conditions. Initially, neural stem cells in sphere-like clusters were induced from human ES (hES cells after being cultured in P-ACM under free-floating conditions. The resultant neural stem cells could circumferentially proliferate under subsequent adhesive culture, and selectively differentiate into neurons or astrocytes by changing the medium to P-ACM or G5, respectively. These hES cell-derived neurons and astrocytes could procure functions similar to those of primary cells. Interestingly, a conditioned medium obtained from the hES cell-derived astrocytes (ES-ACM could successfully be used to substitute P-ACM for induction of neurons. Neurons made by this method could survive in mice brain after xeno-transplantation. CONCLUSION/SIGNIFICANCE: By inducing astrocytes from hES cells in a chemically defined medium, we could produce human neurons without the use of P-ACM. This self-serving method provides an unlimited source of human neural cells and may facilitate clinical applications of hES cells for neurological diseases.

Genetic Tools for Self-Organizing Culture of Mouse Embryonic Stem Cells via Small Regulatory RNA-Mediated Technologies, CRISPR/Cas9, and Inducible RNAi.

Science.gov (United States)

Takata, Nozomu; Sakakura, Eriko; Sakuma, Tetsushi; Yamamoto, Takashi

2017-01-01

Approaches to investigate gene functions in experimental biology are becoming more diverse and reliable. Furthermore, several kinds of tissues and organs that possess their original identities can be generated in petri dishes from stem cells including embryonic, adult and induced pluripotent stem cells. Researchers now have several choices of experimental methods and their combinations to analyze gene functions in various biological systems. Here, as an example we describe one of the better protocols, which combines three-dimensional embryonic stem cell culture with small regulatory RNA-mediated technologies, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), and inducible RNA interference (RNAi). This protocol allows investigation of genes of interest to better understand gene functions in target tissues (or organs) during in vitro development.

Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

Science.gov (United States)

Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

2013-03-01

Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

RNA synthesis in primary cultures of adult rat hepatocytes

International Nuclear Information System (INIS)

Fugassa, E.; Gallo, G.; Voci, A.; Cordone, A.

1983-01-01

The ability of hepatocyte monolayers to synthesize RNA was investigated by measuring [3H]orotic acid incorporation into RNA and the total nuclear RNA polymerase activity as a function of the time in culture. The results demonstrate that primary cultures of hepatocytes maintained in a chemically defined serum- and hormone-free medium are able to synthesize RNA actively. This ability increases within the first 2 d of culture, despite the concomitant decrease in [3H]orotic acid uptake, and decreases only after 3 d. Factors such as serum, insulin, and dexamethasone, known to improve maintenance of functional hepatocytes, markedly stimulate the uptake of labeled precursor without apparently affecting the rate of RNA synthesis by cultured cells. It is suggested that the culture of adult rat hepatocytes provides a useful experimental model for the studies of hormonal regulation of transcription in liver

Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

DEFF Research Database (Denmark)

Meyer, Morten; Widmer, H R; Wagner, B

1998-01-01

days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls...... of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral...... improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar...

Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

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Hertsenberg, Andrew J; Funderburgh, James L

2016-01-01

Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats.

Science.gov (United States)

Matveyenko, Aleksey V; Georgia, Senta; Bhushan, Anil; Butler, Peter C

2010-11-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant.

Co-regulation of the atrial natriuretic factor and cardiac myosin light chain-2 genes during alpha-adrenergic stimulation of neonatal rat ventricular cells. Identification of cis sequences within an embryonic and a constitutive contractile protein gene which mediate inducible expression.

Science.gov (United States)

Knowlton, K U; Baracchini, E; Ross, R S; Harris, A N; Henderson, S A; Evans, S M; Glembotski, C C; Chien, K R

1991-04-25

To study the mechanisms which mediate the transcriptional activation of cardiac genes during alpha adrenergic stimulation, the present study examined the regulated expression of three cardiac genes, a ventricular embryonic gene (atrial natriuretic factor, ANF), a constitutively expressed contractile protein gene (cardiac MLC-2), and a cardiac sodium channel gene. alpha 1-Adrenergic stimulation activates the expression and release of ANF from neonatal ventricular cells. As assessed by RNase protection analyses, treatment with alpha-adrenergic agonists increases the steady-state levels of ANF mRNA by greater than 15-fold. However, a rat cardiac sodium channel gene mRNA is not induced, indicating that alpha-adrenergic stimulation does not lead to an increase in the expression of all cardiac genes. Studies employing a series of rat ANF luciferase and rat MLC-2 luciferase fusion genes identify 315- and 92-base pair cis regulatory sequences within an embryonic gene (ANF) and a constitutively expressed contractile protein gene (MLC-2), respectively, which mediate alpha-adrenergic-inducible gene expression. Transfection of various ANF luciferase reporters into neonatal rat ventricular cells demonstrated that upstream sequences which mediate tissue-specific expression (-3003 to -638) can be segregated from those responsible for inducibility. The lack of inducibility of a cardiac Na+ channel gene, and the segregation of ANF gene sequences which mediate cardiac specific from those which mediate inducible expression, provides further insight into the relationship between muscle-specific and inducible expression during cardiac myocyte hypertrophy. Based on these results, a testable model is proposed for the induction of embryonic cardiac genes and constitutively expressed contractile protein genes and the noninducibility of a subset of cardiac genes during alpha-adrenergic stimulation of neonatal rat ventricular cells.

A procedure for culturing rat neocortex explants in a serum-free nutrient medium

NARCIS (Netherlands)

Romijn, H. J.; de Jong, B. M.; Ruijter, J. M.

1988-01-01

A procedure is described for long-term culturing of rat neocortex explants in a serum-free growth medium. Slices spanning the entire cortical depth from pial to ventricular side are prepared from 6-day-old rat pups. After preincubation in Hanks' balanced salt solution with extra glucose, the

Antitumor Activity of Rat Mesenchymal Stem Cells during Direct or Indirect Co-Culturing with C6 Glioma Cells.

Science.gov (United States)

Gabashvili, A N; Baklaushev, V P; Grinenko, N F; Mel'nikov, P A; Cherepanov, S A; Levinsky, A B; Chehonin, V P

2016-02-01

The tumor-suppressive effect of rat mesenchymal stem cells against low-differentiated rat C6 glioma cells during their direct and indirect co-culturing and during culturing of C6 glioma cells in the medium conditioned by mesenchymal stem cells was studied in an in vitro experiment. The most pronounced antitumor activity of mesenchymal stem cells was observed during direct co-culturing with C6 glioma cells. The number of live C6 glioma cells during indirect co-culturing and during culturing in conditioned medium was slightly higher than during direct co-culturing, but significantly differed from the control (C6 glioma cells cultured in medium conditioned by C6 glioma cells). The cytotoxic effect of medium conditioned by mesenchymal stem cells was not related to medium depletion by glioma cells during their growth. The medium conditioned by other "non-stem" cells (rat astrocytes and fibroblasts) produced no tumor-suppressive effect. Rat mesenchymal stem cells, similar to rat C6 glioma cells express connexin 43, the main astroglial gap junction protein. During co-culturing, mesenchymal stem cells and glioma C6 cells formed functionally active gap junctions. Gap junction blockade with connexon inhibitor carbenoxolone attenuated the antitumor effect observed during direct co-culturing of C6 glioma cells and mesenchymal stem cells to the level produced by conditioned medium. Cell-cell signaling mediated by gap junctions can be a mechanism of the tumor-suppressive effect of mesenchymal stem cells against C6 glioma cells. This phenomenon can be used for the development of new methods of cell therapy for high-grade malignant gliomas.

Effect of D-valine and cytosine arabinoside on [3H]thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

International Nuclear Information System (INIS)

Orgebin-Crist, M.C.; Jonas-Davies, J.; Storey, P.; Olson, G.E.

1984-01-01

Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by [ 3 H]thymidine uptake, is very low. In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured in D-valine medium was significantly lower than that of cells cultured in L-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium. Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures. From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures

[Establishment of sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo].

Science.gov (United States)

Jiang, Hua; Feng, You-Ji; Xie, Yi; Han, Jin-Lan; Wang, Zack; Chen, Tong

2008-10-14

To establish a sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo. Human embryonic stem were (hESCs) were cultured on the mouse embryo fibroblasts and then were induced to differentiate to form three-dimensional EB. The hEBs were cultured in media containing various angiogenesis-related factors: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), endostatin, angiostatin, and platelet factor (PF)-4 of different concentrations for 3 days to observe the sprouting of the hEBs. 3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate labeled acetylated low density lipoprotein (Dil-AcLDL) was added onto the hEBs foe 4 h Immunofluorescence assay was used to observe if Dil-AcLDL was absorbed and if CD31 was expressed so as to determine the existence of embryonic endothelial cells in the sprouting structures. The ideal culturing condition was analyzed. The differentiated EBs formed sprouting structures in the collagen I matrix containing VEGF and FGF. The sprouts among individual EBs were able to link to each other and form vascular network-like structures. In the presence of VEGF and FGF, the sprouts branching from the EBs assimilated Dil-AcLDL, expressed CD31 and formed a 3-dimensional cylindrical organization. The concentrations of growth factors ideally stimulating sprouting growth were 100 ng/ml of VEGF and 50 ng/ml of FGF. The networks among the EBs were abolished by the angiostatin, endostatin, and PF4. The sprouting from hEBs accumulates embryonic endothelial cells and the sprouting network-like structures are indeed endothelial in nature. Inducing of sprouting EBs is an ideal model that mimics early embryonic vasculogenesis in humans.

Radiation effects on pregnant rats. part 1: Morphological changes during pregnancy in rats under effect of gamma rays. Vol. 4

Energy Technology Data Exchange (ETDEWEB)

El-Naggar, M A; Abdel-Wahab, M F; Abdel-Aziz, S M; Abdel-Gawad, I I [Radioisotope Department, Atomic Energy Authority, Cairo (Egypt)

1996-03-01

The following terms were performed to provide a rational systematic understanding of the radiation induced effects on the various stages of embryonic development. The doses delivered were 1, 2, 3, 4 Gy whole body irradiation of the pregnant rats, at specific time periods of gestation. The results obtained are detailed in the text, and supplemented by photographic presentations. Irradiation of pregnant rats on 9{sup th} day of gestation corresponding to placentation stage, and sacrificed on days 14, 18, 21, showed disintegration of embryonic and placental formation on the day 14 which appeared more advanced at higher doses. At later stages of gestation period at days 18 and 21 animals irradiated with low doses showed deformed fetal masses, incompatible with life. At high doses, there was total absence of embryonic and placental formations. Irradiation of pregnant rats on day 13 of gestation corresponding to stage of organogenesis, and sacrificed on days 18 and 21 showed major changes in fetal development. The results obtained are detailed in the text, and supplemented by photographic presentations. 10 figs., 2 tabs.

Radiation effects on pregnant rats. part 1: Morphological changes during pregnancy in rats under effect of gamma rays. Vol. 4

International Nuclear Information System (INIS)

El-Naggar, M.A.; Abdel-Wahab, M.F.; Abdel-Aziz, S.M.; Abdel-Gawad, I.I.

1996-01-01

The following terms were performed to provide a rational systematic understanding of the radiation induced effects on the various stages of embryonic development. The doses delivered were 1, 2, 3, 4 Gy whole body irradiation of the pregnant rats, at specific time periods of gestation. The results obtained are detailed in the text, and supplemented by photographic presentations. Irradiation of pregnant rats on 9 th day of gestation corresponding to placentation stage, and sacrificed on days 14, 18, 21, showed disintegration of embryonic and placental formation on the day 14 which appeared more advanced at higher doses. At later stages of gestation period at days 18 and 21 animals irradiated with low doses showed deformed fetal masses, incompatible with life. At high doses, there was total absence of embryonic and placental formations. Irradiation of pregnant rats on day 13 of gestation corresponding to stage of organogenesis, and sacrificed on days 18 and 21 showed major changes in fetal development. The results obtained are detailed in the text, and supplemented by photographic presentations. 10 figs., 2 tabs

Radiation-induced PKC signaling system in cultured rat hepatocytes

International Nuclear Information System (INIS)

Nakajima, Tetsuo; Yukawa, Osami

1998-01-01

Radiation effects on living organisms are mainly caused through reactive oxygen species (ROS) on living cells. It is known that ROS damages various membranes and the bio membranes play an important role in cellular signal transduction pathways. The effects of radiation on cellular signal transduction pathways in cultured rat hepatocytes have been studied

A comparison of the embryonic stem cell test and whole embryo culture assay combined with the BeWo placental passage model for predicting the embryotoxicity of azoles.

NARCIS (Netherlands)

Dimopoulou, Myrto; Verhoef, Aart; Gomes, Caroline A; van Dongen, Catharina W; Rietjens, Ivonne M C M; Piersma, Aldert H; van Ravenzwaay, Bennard

2018-01-01

In the present study, we show the value of combining toxico-dynamic and -kinetic in vitro approaches for embryotoxicity testing of azoles. Both the whole embryo culture (WEC) and the embryonic stem cells test (EST) predicted the in vivo potency ranking of twelve tested azoles with moderate accuracy.

Directional differentiation of chicken embryonic stem cells into ...

African Journals Online (AJOL)

Chicken embryonic stem (ES) cells are useful for producing transgenic chickens and preserving genetic material in avian species. In this study, the differentiation potential of chicken ES cells was investigated in vitro. Chicken ES cells were differentiated into osteoblasts cultured for 15 to 21 days in the induction media ...

Effect of hypertensive rat plasma on ion transport of cultured vascular smooth muscle

International Nuclear Information System (INIS)

Magargal, W.W.; Overbeck, H.W.

1986-01-01

We layered fresh, unprocessed plasma from healthy rats with early (less than or equal to 7 days) or benign, chronic (greater than 3 wk) one-kidney, one-clip hypertension and from paired one-kidney normotensive control rats over confluent primary-cultured rat aortic smooth muscle cells. Plasma from all rats increased cellular ouabain-sensitive 86 Rb + uptake and sodium content and decreased ouabain-insensitive 86 Rb + uptake compared with uptakes and content in the presence of balanced salt solution (P less than 0.01). Cells incubated in the presence of plasma from rats with early (P less than 0.02) or chronic hypertension (P less than 0.01) had significantly reduced ouabain-sensitive 86 Rb + uptake when compared with cells incubated in normotensive plasma, but their intracellular Na+ contents were not lower. We no longer detected this uptake difference when chronic hypertensives drank 0.9% NaCl instead of water. Plasma from hypertensive rats also altered ouabain-insensitive 86 Rb + uptake by the cultured cells. These findings of this new, reproducible, and specific assay system support the hypothesis that plasma factors inhibit the membrane sodium-potassium pump in vascular smooth muscle cells in this form of hypertension. The abnormality occurs in both early and chronic stages, but may not be related to sodium intake. The data also provide evidence for plasma factors in hypertension altering membrane K+ permeability

Activation of the transcription factor carbohydrate-responsive element-binding protein by glucose leads to increased pancreatic beta cell differentiation in rats.

Science.gov (United States)

Soggia, A; Flosseau, K; Ravassard, P; Szinnai, G; Scharfmann, R; Guillemain, G

2012-10-01

Pancreatic cell development is a tightly controlled process. Although information is available regarding the mesodermal signals that control pancreatic development, little is known about the role of environmental factors such as nutrients, including glucose, on pancreatic development. We previously showed that glucose and its metabolism through the hexosamine biosynthesis pathway (HBP) promote pancreatic endocrine cell differentiation. Here, we analysed the role of the transcription factor carbohydrate-responsive element-binding protein (ChREBP) in this process. This transcription factor is activated by glucose, and has been recently described as a target of the HBP. We used an in vitro bioassay in which pancreatic endocrine and exocrine cells develop from rat embryonic pancreas in a way that mimics in vivo pancreatic development. Using this model, gain-of-function and loss-of-function experiments were undertaken. ChREBP was produced in the endocrine lineage during pancreatic development, its abundance increasing with differentiation. When rat embryonic pancreases were cultured in the presence of glucose or xylitol, the production of ChREBP targets was induced. Concomitantly, beta cell differentiation was enhanced. On the other hand, when embryonic pancreases were cultured with inhibitors decreasing ChREBP activity or an adenovirus producing a dominant-negative ChREBP, beta cell differentiation was reduced, indicating that ChREBP activity was necessary for proper beta cell differentiation. Interestingly, adenovirus producing a dominant-negative ChREBP also reduced the positive effect of N-acetylglucosamine, a substrate of the HBP acting on beta cell differentiation. Our work supports the idea that glucose, through the transcription factor ChREBP, controls beta cell differentiation from pancreatic progenitors.

Development of human nervous tissue upon differentiation of embryonic stem cells in three-dimensional culture.

Science.gov (United States)

Preynat-Seauve, Olivier; Suter, David M; Tirefort, Diderik; Turchi, Laurent; Virolle, Thierry; Chneiweiss, Herve; Foti, Michelangelo; Lobrinus, Johannes-Alexander; Stoppini, Luc; Feki, Anis; Dubois-Dauphin, Michel; Krause, Karl Heinz

2009-03-01

Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air-liquid interface-based culture of human ESC. This culture system allows three-dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3-month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural-like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air-liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural-like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three-dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues.

Increased adipogenesis in cultured embryonic chondrocytes and in adult bone marrow of dominant negative Erg transgenic mice.

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Sébastien Flajollet

Full Text Available In monolayer culture, primary articular chondrocytes have an intrinsic tendency to lose their phenotype during expansion. The molecular events underlying this chondrocyte dedifferentiation are still largely unknown. Several transcription factors are important for chondrocyte differentiation. The Ets transcription factor family may be involved in skeletal development. One family member, the Erg gene, is mainly expressed during cartilage formation. To further investigate the potential role of Erg in the maintenance of the chondrocyte phenotype, we isolated and cultured chondrocytes from the rib cartilage of embryos of transgenic mice that express a dominant negative form of Erg (DN-Erg during cartilage formation. DN-Erg expression in chondrocytes cultured for up to 20 days did not affect the early dedifferentiation usually observed in cultured chondrocytes. However, lipid droplets accumulated in DN-Erg chondrocytes, suggesting adipocyte emergence. Transcriptomic analysis using a DNA microarray, validated by quantitative RT-PCR, revealed strong differential gene expression, with a decrease in chondrogenesis-related markers and an increase in adipogenesis-related gene expression in cultured DN-Erg chondrocytes. These results indicate that Erg is involved in either maintaining the chondrogenic phenotype in vitro or in cell fate orientation. Along with the in vitro studies, we compared adipocyte presence in wild-type and transgenic mice skeletons. Histological investigations revealed an increase in the number of adipocytes in the bone marrow of adult DN-Erg mice even though no adipocytes were detected in embryonic cartilage or bone. These findings suggest that the Ets transcription factor family may contribute to the homeostatic balance in skeleton cell plasticity.

Islet graft survival and function: concomitant culture and transplantation with vascular endothelial cells in diabetic rats.

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Pan, Xiaoming; Xue, Wujun; Li, Yang; Feng, Xinshun; Tian, Xiaohui; Ding, Chenguang

2011-12-15

Human islet transplantation is a great potential therapy for type I diabetes. To investigate islet graft survival and function, we recently showed the improved effects after co-culture and co-transplantation with vascular endothelial cells (ECs) in diabetic rats. ECs were isolated, and the viability of isolated islets was assessed in two groups (standard culture group and co-culture group with ECs). Then streptozotocin-induced diabetic rats were divided into four groups before islet transplantation as follows: group A with infusion of islet grafts; group B with combined vascular ECs and islet grafts; groups C and D as controls with single ECs infusion and phosphate-buffered saline injection, respectively. Blood glucose and insulin concentrations were measured daily. Expression of vascular endothelial growth factor was investigated by immunohistochemical staining. The mean microvascular density was also calculated. More than 90% of acridine orange-propidium iodide staining positive islets demonstrated normal morphology while co-cultured with ECs for 7 days. Compared with standard control, insulin release assays showed a significantly higher simulation index in co-culture group except for the first day (Ptransplantation, there was a significant difference in concentrations of blood glucose and insulin among these groups after 3 days (Pislet group (P=0.04). Co-culture with ECs in vitro could improve the survival and function of isolated rat islet, and co-transplantation of islets with ECs could effectively prolong the islet graft survival in diabetic rats.

The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension

DEFF Research Database (Denmark)

Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E

2010-01-01

Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts t...

Control of fibronectin synthesis by rat granulosa cells in culture

International Nuclear Information System (INIS)

Skinner, M.K.; Dorrington, J.H.

1984-01-01

The secreted and cellular [ 35 S]methionine-radiolabeled proteins of cultured rat granulosa cells were separated by electrophoresis on sodium dodecylsulfate (SDS) polyacrylamide gradient slab gels. From 24 to 72 h of culture FSH increased the intensity of labeling of most of the secreted proteins. A 220,000-dalton protein, however, increased in intensity only in control cultures and became the major secreted protein after 72 h, comprising 20% of the total radiolabeled proteins. This protein was identified as fibronectin by immunoprecipitation. There was no increase in the secreted or cellular fibronectin in FSH- or testosterone- and insulin-treated cultures. These studies indicate that a component of extracellular matrix is a major secretory product of unstimulated immature granulosa cells. As hormones induce the differentiated functions of granulosa cells in culture, the secretion of fibronectin is inhibited

Generation of stomach tissue from mouse embryonic stem cells.

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Noguchi, Taka-aki K; Ninomiya, Naoto; Sekine, Mari; Komazaki, Shinji; Wang, Pi-Chao; Asashima, Makoto; Kurisaki, Akira

2015-08-01

Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, β-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.

Royal Jelly Prevents Osteoporosis in Rats: Beneficial Effects in Ovariectomy Model and in Bone Tissue Culture Model

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Saburo Hidaka

2006-01-01

Full Text Available Royal jelly (RJ has been used worldwide for many years as medical products, health foods and cosmetics. Since RJ contains testosterone and has steroid hormone-type activities, we hypothesized that it may have beneficial effects on osteoporosis. We used both an ovariectomized rat model and a tissue culture model. Rats were divided into eight groups as follows: sham-operated (Sham, ovariectomized (OVX, OVX given 0.5% (w/w raw RJ, OVX given 2.0% (w/w RJ, OVX given 0.5% (w/w protease-treated RJ (pRJ, OVX given 2.0% (w/w pRJ, OVX given 17β-estradiol and OVX given its vehicle, respectively. The Ovariectomy decreased tibial bone mineral density (BMD by 24%. Administration of 17β-estradiol to OVX rats recovered the tibial BMD decrease by 100%. Administration of 2.0% (w/w RJ and 0.5–2.0% (w/w pRJ to OVX rats recovered it by 85% or more. These results indicate that both RJ and pRJ are almost as effective as 17β-estradiol in preventing the development of bone loss induced by ovariectomy in rats. In tissue culture models, both RJ and pRJ increased calcium contents in femoral-diaphyseal and femoral-metaphyseal tissue cultures obtained from normal male rats. However, in a mouse marrow culture model, they neither inhibited the parathyroid hormone (PTH-induced calcium loss nor affected the formation of osteoclast-like cells induced by PTH in mouse marrow culture system. Therefore, our results suggest that both RJ and pRJ may prevent osteoporosis by enhancing intestinal calcium absorption, but not by directly antagonizing the action of PTH.

Function of JARID2 in bovines during early embryonic development

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Yao Fu

2017-12-01

efficiency of in vitro embryo culture as well as a theoretical basis for further studying the regulatory mechanisms involved in early embryonic development.

EVA1A/TMEM166 Regulates Embryonic Neurogenesis by Autophagy

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Mengtao Li

2016-03-01

Full Text Available Self-renewal and differentiation of neural stem cells is essential for embryonic neurogenesis, which is associated with cell autophagy. However, the mechanism by which autophagy regulates neurogenesis remains undefined. Here, we show that Eva1a/Tmem166, an autophagy-related gene, regulates neural stem cell self-renewal and differentiation. Eva1a depletion impaired the generation of newborn neurons, both in vivo and in vitro. Conversely, overexpression of EVA1A enhanced newborn neuron generation and maturation. Moreover, Eva1a depletion activated the PIK3CA-AKT axis, leading to the activation of the mammalian target of rapamycin and the subsequent inhibition of autophagy. Furthermore, addition of methylpyruvate to the culture during neural stem cell differentiation rescued the defective embryonic neurogenesis induced by Eva1a depletion, suggesting that energy availability is a significant factor in embryonic neurogenesis. Collectively, these data demonstrated that EVA1A regulates embryonic neurogenesis by modulating autophagy. Our results have potential implications for understanding the pathogenesis of neurodevelopmental disorders caused by autophagy dysregulation.

The output of neuronotrophic and neurite-promoting agents from rat brain astroglial cells: a microculture method for screening potential regulatory molecules.

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Rudge, J S; Manthorpe, M; Varon, S

1985-04-01

Throughout embryonic development, as well as in response to injury of the central nervous system, astroglial cells may present neurons with a critical supply of neuronotrophic and neurite-promoting factors which control, respectively, neuronal survival and axonal growth. The identification of such astroglial cell-derived factors, as well as of specific extrinsic agents regulating their production, will require the use of in vitro techniques. We define here a new microculture system in which added agents can be screened for their ability to enhance or inhibit the output of trophic and neurite-promoting factors from purified neonatal rat brain astroglial cells. With such a procedure, thousands of replicate secondary astroglial cultures can be set-up and maintained in chemically defined medium, on a defined substratum and in a viable, low proliferative stable state. These cultured astroglial cells release into their medium at least three distinct and separable types of agents addressing nerve cells in vitro: (i) high molecular weight trophic factors (Mr greater than 10,000) which support the survival of embryonic peripheral neurons; (ii) low molecular weight trophic agents (Mr less than 10,000) supporting embryonic central neurons; and (iii) polyornithine-binding neurite-promoting factors which enhance neuritic regeneration for both peripheral and central neurons. The temporal release patterns of these three agents from astroglial cultures are quite distinct suggesting that their output is independently regulated.

Rotary orbital suspension culture of embryonic stem cell-derived neural stem/progenitor cells: impact of hydrodynamic culture on aggregate yield, morphology and cell phenotype.

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Laundos, Tiago L; Silva, Joana; Assunção, Marisa; Quelhas, Pedro; Monteiro, Cátia; Oliveira, Carla; Oliveira, Maria J; Pêgo, Ana P; Amaral, Isabel F

2017-08-01

Embryonic stem (ES)-derived neural stem/progenitor cells (ES-NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single-cell suspensions of ES-NSPCs, to obtain homogeneously-sized ES-NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES-NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller-sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES-NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N-cadherin and β-catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase-2 (MMP-2) activity and percentage of neuronal cells. In line with the increased MMP-2 activity levels found, hydrodynamically-cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single-cell suspensions of ES-NSPCs, besides allowing the formation of homogeneously-sized neurospheres, promoted ES-NSPC differentiation and outward migration, possibly by influencing the expression of cell-cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly-sized ES-NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or

Derivation of mouse embryonic stem cell lines from tyrosine hydroxylase reporter mice crossed with a human SNCA transgenic mouse model of Parkinson's disease

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Margarita Chumarina

2017-03-01

Full Text Available Mouse embryonic stem cell (mESC lines were derived by crossing heterozygous transgenic (tg mice expressing green fluorescent protein (GFP under the control of the rat tyrosine hydroxylase (TH promoter, with homozygous alpha-synuclein (aSYN mice expressing human mutant SNCAA53T under the control of the mouse Prion promoter (MoPrP, or wildtype (WT mice. The expression of GFP and human aSYN was validated by immunocytochemistry in midbrain neuron cultures upon differentiation of mESC lines using stromal cell-derived inducing activity. These mESC lines can help to study the impact of human aSYN expression in neurons and oligodendrocytes, and also trace GFP-expressing midbrain neurons.

Partly ordered synthesis and degradation of glycogen in cultured rat myotubes

DEFF Research Database (Denmark)

Elsner, Peter; Quistorff, Bjørn; Hansen, Gert H

2001-01-01

The following questions concerning glycogen synthesis and degradation were examined in cultured rat myotubes. 1) Is synthesis and degradation of the individual glycogen molecule a strictly ordered process, with the last glucosyl unit incorporated into the molecule being the first to be released...

Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment.

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Yoon, Byung Sun; Yoo, Seung Jun; Lee, Jeoung Eun; You, Seungkwon; Lee, Hoon Taek; Yoon, Hyun Soo

2006-04-01

Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is, however, challenged by a limited supply of appropriate cells. Therefore, we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study, we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine, a well-known demethylating agent, and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1, 1, or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion, these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes.

Effect of various chemicals on the metabolism of benzo(a)pyrene by cultured rat colon

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Autrup, Herman; Harris, Curtis C.; Fugaro, Steven

1977-01-01

The effect of various co- and anti-carcinogens of colon carcinogenesis on the metabolism of benzo(a)pyrene (BP) in cultured rat colon is reported. Rat colon enzymatically converted BP into metabolites which bind to cellular macromolecules i.e., DNA and protein. Activity of aryl hydrocarbon...

AlGaN/GaN-based HEMTs for electrical stimulation of neuronal cell cultures

International Nuclear Information System (INIS)

Witte, H; Warnke, C; Krost, A; Voigt, T; De Lima, A; Ivanov, I; Vidakovic-Koch, T R; Sundmacher, K

2011-01-01

Unipolar source-drain voltage pulses of GaN/AlGaN-high electron mobility transistors (HEMTs) were used for stimulation of cultured neuronal networks obtained from embryonic rat cerebral cortex. The HEMT sensor was grown by metal organic vapour phase epitaxy on a 2 inch sapphire substrate consisting of 10 single HEMTs concentrically arranged around the wafer centre. Electrolytic reactions between the HEMT sensor surface and the culture medium were not detected using cyclic voltammetry. During voltage pulses and resulting neuronal excitation, capacitances were recharged giving indications of the contributions of the AlGaN and AlO x isolation layers between the two-dimensional electron gas channel and the neuron culture. The resulting threshold current for stimulation of neuron activity strongly depended on the culture and HEMT position on the sensor surface under consideration which was caused by different impedances of each neuron culture and position within the culture. The differences of culture impedances could be explained by variations of composition, thickness and conductivity of the culture areas.

AlGaN/GaN-based HEMTs for electrical stimulation of neuronal cell cultures

Energy Technology Data Exchange (ETDEWEB)

Witte, H; Warnke, C; Krost, A [Institute of Experimental Physics, Otto-von-Guericke-University-Magdeburg, Magdeburg (Germany); Voigt, T; De Lima, A [Institute for Physiology, Otto-von-Guericke-University-Magdeburg, Magdeburg (Germany); Ivanov, I; Vidakovic-Koch, T R; Sundmacher, K, E-mail: hartmut.witte@physik.uni-magdeburg.de [Process Systems Engineering, Otto-von-Guericke-University-Magdeburg, Magdeburg (Germany)

2011-09-07

Unipolar source-drain voltage pulses of GaN/AlGaN-high electron mobility transistors (HEMTs) were used for stimulation of cultured neuronal networks obtained from embryonic rat cerebral cortex. The HEMT sensor was grown by metal organic vapour phase epitaxy on a 2 inch sapphire substrate consisting of 10 single HEMTs concentrically arranged around the wafer centre. Electrolytic reactions between the HEMT sensor surface and the culture medium were not detected using cyclic voltammetry. During voltage pulses and resulting neuronal excitation, capacitances were recharged giving indications of the contributions of the AlGaN and AlO{sub x} isolation layers between the two-dimensional electron gas channel and the neuron culture. The resulting threshold current for stimulation of neuron activity strongly depended on the culture and HEMT position on the sensor surface under consideration which was caused by different impedances of each neuron culture and position within the culture. The differences of culture impedances could be explained by variations of composition, thickness and conductivity of the culture areas.

Protective Effect of Edaravone against Carbon Monoxide Induced Apoptosis in Rat Primary Cultured Astrocytes

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Xiaodan Xu

2017-01-01

Full Text Available Objective. To observe the protective effect of edaravone (Eda on astrocytes after prolonged exposure to carbon monoxide (CO and further to investigate the potential mechanisms of Eda against CO-induced apoptosis. Methods. The rat primary cultured astrocytes were cultured in vitro and exposed to 1% CO for 24 h after being cultured with different concentrations of Eda. MTT assay was used to detect the cytotoxicity of CO. Flow cytometry was used to detect the apoptosis rate, membrane potential of mitochondria, and ROS level. The mRNA and protein expressions of Bcl-2, Bax, and caspase-3 were assessed by real-time PCR and Western blotting analysis, respectively. Results. Eda can significantly suppress cytotoxicity of CO, and it can significantly increase membrane potential of mitochondria and Bcl-2 expressions and significantly suppress the apoptosis rate, ROS level, Bax, and caspase-3 expressions. Conclusion. Eda protects against CO-induced apoptosis in rat primary cultured astrocytes through decreasing ROS production and subsequently inhibiting mitochondrial apoptosis pathway.

Dopaminergic differentiation of human neural stem cells mediated by co-cultured rat striatal brain slices

DEFF Research Database (Denmark)

Anwar, Mohammad Raffaqat; Andreasen, Christian Maaløv; Lippert, Solvej Kølvraa

2008-01-01

differentiation, we co-cultured cells from a human neural forebrain-derived stem cell line (hNS1) with rat striatal brain slices. In brief, coronal slices of neonatal rat striatum were cultured on semiporous membrane inserts placed in six-well trays overlying monolayers of hNS1 cells. After 12 days of co......Properly committed neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. To establish a setting for identification of secreted neural compounds promoting dopaminergic...

Expression pattern of pluripotent markers in different embryonic developmental stages of buffalo (Bubalus bubalis) embryos and putative embryonic stem cells generated by parthenogenetic activation.

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Singh, Karn P; Kaushik, Ramakant; Garg, Veena; Sharma, Ruchi; George, Aman; Singh, Manoj K; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K; Chauhan, Manmohan S

2012-12-01

In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.

L1TD1 Is a Marker for Undifferentiated Human Embryonic Stem Cells

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Wong, Raymond Ching-Bong; Ibrahim, Abel; Fong, Helen; Thompson, Noelle; Lock, Leslie F.; Donovan, Peter J.

2011-01-01

Background Human embryonic stem cells (hESC) are stem cells capable of differentiating into cells representative of the three primary embryonic germ layers. There has been considerable interest in understanding the mechanisms regulating stem cell pluripotency, which will ultimately lead to development of more efficient methods to derive and culture hESC. In particular, Oct4, Sox2 and Nanog are transcription factors known to be important in maintenance of hESC. However, many of the downstream ...

Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells

DEFF Research Database (Denmark)

Anisimov, Sergey V.; Christophersen, Nicolaj S.; Correia, Ana S.

2011-01-01

The majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both...... the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early...... foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates....

Real-time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device.

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Super, Alexandre; Jaccard, Nicolas; Cardoso Marques, Marco Paulo; Macown, Rhys Jarred; Griffin, Lewis Donald; Veraitch, Farlan Singh; Szita, Nicolas

2016-09-01

Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real-time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time-course data for bulk and peri-cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non-invasive and label-free approach. Additionally, we confirmed non-invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell(-1) s(-1) , and 5 and 35 amol cell(-1) s(-1) were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non-invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell-based therapies. © 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cultured rat and purified human Pneumocystis carinii stimulate intra- but not extracellular free radical production in human neutrophils

DEFF Research Database (Denmark)

Jensen, T; Aliouat, E M; Lundgren, B

1998-01-01

The production of free radicals in human neutrophils was studied in both Pneumocystis carinii derived from cultures of L2 rat lung epithelial-like cells and Pneumocystis carinii purified from human lung. Using the cytochrome C technique, which selectively measured extracellular superoxide...... generation, hardly any free radical production was observed after stimulation with cultured rat-derived P. carinii. A chemiluminescence technique, which separately measured intra- and extracellular free radical production, was subsequently employed to differentiate the free radical generation....... It was established that 1) P. carinii stimulated intra- but not extracellular free radical production in human neutrophils, 2) opsonized cultured rat-derived P. carinii stimulated human neutrophils to a strong intracellular response of superoxide production, and 3) opsonized P. carinii, purified from human lung also...

Impairment of male reproduction in adult rats exposed to hydroxyprogesterone caproate in utero

Science.gov (United States)

Pushpalatha, T.; Ramachandra Reddy, P.; Sreenivasula Reddy, P.

Hydroxyprogesterone caproate is one of the most effective and widely used drugs for the treatment of uterine bleeding and threatened miscarriage in women. Hydroxyprogesterone caproate was administered to pregnant rats in order to assess the effect of intraperitoneal exposure to supranormal levels of hydroxyprogesterone caproate on the male reproductive potential in the first generation. The cauda epididymal sperm count and motility decreased significantly in rats exposed to hydroxyprogesterone caproate during embryonic development, when compared with control rats. The levels of serum testosterone decreased with an increase in follicle stimulating hormone and luteinizing hormone in adult rats exposed to hydroxyprogesterone caproate during the embryonic stage. It was suggested that the impairment of male reproductive performance could be mediated through the inhibition of testosterone production.

Differential feedback regulation of cholesterol 7α-hydroxylase mRNA and transcriptional activity by rat bile acids in primary monolayer cultures of rat hepatocytes

NARCIS (Netherlands)

Twisk, J.; Lehmann, E.M.; Princen, H.M.G.

1993-01-01

We have used primary monolayer cultures of rat hepatocytes to study the effects of physiological concentrations of various bile acids, commonly found in bile of normal rats, on the mechanism of regulation of cholesterol 7α-hydroxylase and bile acid synthesis. Addition of taurocholic acid, the most

Protective effects of resveratrol on ethanol-induced apoptosis in embryonic stem cells and disruption of embryonic development in mouse blastocysts

International Nuclear Information System (INIS)

Huang, L.-H.; Shiao, N.-H.; Hsuuw, Y.-D.; Chan, W.-H.

2007-01-01

Previous studies have established that ethanol induces apoptosis, but the precise molecular mechanisms are currently unclear. Here, we show that 0.3-1.0% (w/v) ethanol induces apoptosis in mouse blastocysts and that resveratrol, a grape-derived phytoalexin with known antioxidant and anti-inflammatory properties, prevents ethanol-induced apoptosis and inhibition of cell proliferation. Moreover, ethanol-treated blastocysts show normal levels of implantation on culture dishes in vitro but a reduced ability to reach the later stages of embryonic development. Pretreatment with resveratrol prevented ethanol-induced disruption of embryonic development in vitro and in vivo. In an in vitro cell-based assay, we further found that ethanol increases the production of reactive oxygen species in ESC-B5 embryonic stem cells, leading to an increase in the intracellular concentrations of cytoplasmic free Ca 2+ and NO, loss of mitochondrial membrane potential, mitochondrial release of cytochrome c, activation of caspase-9 and -3, and apoptosis. These changes were blocked by pretreatment with resveratrol. Based on these results, we propose a model for the protective effect of resveratrol on ethanol-induced cell injury in blastocysts and ESC-B5 cells

Cell structure and proliferative activity of organ cultures of normal embryonic lung tissue of mice resistant (C57BL) and predisposed (A) to lung tumors

International Nuclear Information System (INIS)

Kolesnichenko, T.S.; Gor'kova, T.G.

1985-01-01

Local factors such as proliferative activity and the numerical ratio between epithelial and mesenchymal cells, and also the character of interaction between the tissue components in ontogeny may play an important role in the realization of sensitivity of mice of a particular line to the development of lung tumors. These characteristics of lung tissue in mice of lines A and C57BL are investigated under normal conditions and during induced carcinogenesis. Results are given of a comparative study of the relative numbers of epithelial and mesenchymal cells in organ cultures of embryonic lungs. 3 H-thymidine was added to the cultures on the 14th day of the experiment in a concentration of 1 microCi/m1 medium. An autoradiographic study of the cultures was performed

Mechanical signaling coordinates the embryonic heartbeat

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Chiou, Kevin K.; Rocks, Jason W.; Chen, Christina Yingxian; Cho, Sangkyun; Merkus, Koen E.; Rajaratnam, Anjali; Robison, Patrick; Tewari, Manorama; Vogel, Kenneth; Majkut, Stephanie F.; Prosser, Benjamin L.; Discher, Dennis E.; Liu, Andrea J.

2016-01-01

In the beating heart, cardiac myocytes (CMs) contract in a coordinated fashion, generating contractile wave fronts that propagate through the heart with each beat. Coordinating this wave front requires fast and robust signaling mechanisms between CMs. The primary signaling mechanism has long been identified as electrical: gap junctions conduct ions between CMs, triggering membrane depolarization, intracellular calcium release, and actomyosin contraction. In contrast, we propose here that, in the early embryonic heart tube, the signaling mechanism coordinating beats is mechanical rather than electrical. We present a simple biophysical model in which CMs are mechanically excitable inclusions embedded within the extracellular matrix (ECM), modeled as an elastic-fluid biphasic material. Our model predicts strong stiffness dependence in both the heartbeat velocity and strain in isolated hearts, as well as the strain for a hydrogel-cultured CM, in quantitative agreement with recent experiments. We challenge our model with experiments disrupting electrical conduction by perfusing intact adult and embryonic hearts with a gap junction blocker, β-glycyrrhetinic acid (BGA). We find this treatment causes rapid failure in adult hearts but not embryonic hearts—consistent with our hypothesis. Last, our model predicts a minimum matrix stiffness necessary to propagate a mechanically coordinated wave front. The predicted value is in accord with our stiffness measurements at the onset of beating, suggesting that mechanical signaling may initiate the very first heartbeats. PMID:27457951

[Lessening effect of hypoxia-preconditioned rat cerebrospinal fluid on oxygen-glucose deprivation-induced injury of cultured hippocampal neurons in neonate rats and possible mechanism].

Science.gov (United States)

Niu, Jing-Zhong; Zhang, Yan-Bo; Li, Mei-Yi; Liu, Li-Li

2011-12-25

The present study was to investigate the effect of cerebrospinal fluid (CSF) from the rats with hypoxic preconditioning (HPC) on apoptosis of cultured hippocampal neurons in neonate rats under oxygen glucose deprivation (OGD). Adult Wistar rats were exposed to 3 h of hypoxia for HPC, and then their CSF was taken out. Cultured hippocampal neurons from the neonate rats were randomly divided into four groups (n = 6): normal control group, OGD group, normal CSF group and HPC CSF group. OGD group received 1.5 h of incubation in glucose-free Earle's solution containing 1 mmol/L Na2S2O4, and normal and HPC CSF groups were subjected to 1 d of corresponding CSF treatments followed by 1.5 h OGD. The apoptosis of neurons was analyzed by confocal laser scanning microscope and flow cytometry using Annexin V/PI double staining. Moreover, protein expressions of Bcl-2 and Bax were detected by immunofluorescence. The results showed that few apoptotic cells were observed in normal control group, whereas the number of apoptotic cells was greatly increased in OGD group. Both normal and HPC CSF could decrease the apoptosis of cultured hippocampal neurons injured by OGD (P neurons by up-regulating expression of Bcl-2 and down-regulating expression of Bax.

Epigenetic stability, adaptability, and reversibility in human embryonic stem cells

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Tompkins, Joshua D.; Hall, Christine; Chen, Vincent Chang-yi; Li, Arthur Xuejun; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

2012-01-01

The stability of human embryonic stem cells (hESCs) is of critical importance for both experimental and clinical applications. We find that as an initial response to altered culture conditions, hESCs change their transcription profile for hundreds of genes and their DNA methylation profiles for several genes outside the core pluripotency network. After adaption to conditions of feeder-free defined and/or xeno-free culture systems, expression and DNA methylation profiles are quite stable for a...

Effects of Treatment with Bone Morphogenetic Protein 4 and Co-culture on Expression of Piwil2 Gene in Mouse Differentiated Embryonic Stem Cells

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Mehdi Forouzandeh-Moghadam

2009-01-01

Full Text Available Background: Specific growth factors and feeder layers seem to have important roles in in vitroembryonic stem cells (ESCs differentiation. In this study,the effects of bone morphogenetic protein4 (BMP4 and mouse embryonic fibroblasts (MEFs co-culture system on germ cell differentiationfrom mouse ESCs were studied.Materials and Methods: Cell suspension was prepared from one-day-old embryoid body (EBand cultured for four days in DMEM medium containing 20% fetal bovine serum (FBS in thefollowing groups: simple culture (SC, simple culture with BMP4 (SCB, co-culture (CO-C andco-culture with BMP4 (CO-CB. Expression of piwi-like homolog 2 (Piwil2, the germ cell-specificgene, was evaluated in the different study groups by using quantitative real time polymerase chainreaction (RT-PCR. Testis was used as a positive control.Results: The maximum and minimum Piwil2 expression was observed in SC and SCB groups,respectively. A significant difference was observed in Piwil2 expression between SCB and otherstudy groups (p<0.05.Conclusion: The findings of this study showed that neither the addition of BMP4 in culture mediumnor the use of MEFs as a feeder layer have a positive effect on late germ cell induction from mouseESCs.

Use of 5-Bromodeoxyuridine and irradiation for the estimation of the myoblast and myocyte content of primary rat heart cell cultures

International Nuclear Information System (INIS)

Masse, M.J.O.; Harary, I.

1980-01-01

A method for killing dividing cells was adapted for the elimination of dividing heart muscle cells (myoblasts) in cultures. We have used this method to demonstrate their presence and to estimate their number as well as the number of nondividing heart muscle cells (myocytes) in the neo-natal rat heart. Cells were cultivated in BUdR (5-bromodeoxyuridine) 10 -4 M for 3 days and then irradiated with long uv light. The selective elimination of dividing cells led to a loss of myosin Ca 2+ -activated ATPase in the cultures. The percent of ATPase left after irradiation was 32% of the control in cultures derived from 1-day postnatal rats and 48% in cultures from 4-day postnatal rats. This reflects an in vivo shift of myoblasts to myocytes in the muscle cell population as the rat ages

Characteristics of monolayer culture of bone marrow cells of rats bearing 239Pu-induced osteosarcoma

International Nuclear Information System (INIS)

Bukhtoyarova, Z.M.; Lemberg, V.K.

1984-01-01

The report is concerned with a monolayer culture of bone marrow cells of rats in which optimal blastogenic dose (92.5 kBq/kg) induced osteosarcoma. The cell culture showed an enhanced rate of fibroblast-like cell proliferation (increased number of mitoses and symplasts and larger colonies of cells), apparent signs of radiation in ury (pathologic mitoses, chromosome aberrations and gaps) as well as an increase in ploidy. Diffusion chamber measurements demonstrated osteogenic precursor-cells in osteosarcoma-bearing rats to be highly capable of bone formation. This relatively high ability seems to occur outside bone marrow as well

Functional characterization of apical transporters expressed in rat proximal tubular cells (PTCs) in primary culture.

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Nakanishi, Takeo; Fukushi, Akimasa; Sato, Masanobu; Yoshifuji, Mayuko; Gose, Tomoka; Shirasaka, Yoshiyuki; Ohe, Kazuyo; Kobayashi, Masato; Kawai, Keiichi; Tamai, Ikumi

2011-12-05

Since in vitro cell culture models often show altered apical transporter expression, they are not necessarily suitable for the analysis of renal transport processes. Therefore, we aimed here to investigate the usefulness of primary-cultured rat proximal tubular cells (PTCs) for this purpose. After isolation of renal cortical cells from rat kidneys, PTCs were enriched and the gene expression and function of apical transporters were analyzed by means of microarray, RT-PCR and uptake experiments. RT-PCR confirmed that the major apical transporters were expressed in rat PTCs. Na(+)-dependent uptake of α-methyl-d-glucopyranoside (αMG), ergothioneine and carnitine by the PTCs suggests functional expression of Sglts, Octn1 and Octn2, respectively. Inhibition of pH-dependent glycylsarcosine uptake by low concentration of cephalexin, which is a β-lactam antibiotics recognized by Pepts, indicates a predominant role of high affinity type Pept2, but not low affinity type Pept1, in the PTCs. Moreover, the permeability ratio of [(14)C]αMG (apical to basolateral/basolateral to apical) across PTCs was 4.3, suggesting that Sglt-mediated reabsorptive transport is characterized. In conclusion, our results indicate that rat PTCs in primary culture are found to be a promising in vitro model to evaluate reabsorption processes mediated at least by Sglts, Pept2, Octn1 and Octn2.

Hepatoprotective effects of Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] on alcohol-damaged primary rat hepatocyte culture in vitro.

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Jiang, Wenhua; Bian, Yuzhu; Wang, Zhenghui; Chang, Thomas Ming Swi

2017-02-01

We have prepared a novel nanobiotherapeutic, Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase], which not only transports both oxygen and carbon dioxide but also a therapeutic antioxidant. Our previous study in a severe sustained 90 min hemorrhagic shock rat model shows that it has a hepatoprotective effect. We investigate its hepatoprotective effect further in this present report using an alcohol-damaged primary hepatocyte culture model. Results show that it significantly reduced ethanol-induced AST release, lipid peroxidation, and ROS production in rat primary hepatocytes culture. It also significantly enhanced the viability of ethanol-treated hepatocytes. Thus, the result shows that Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] also has some hepatoprotective effects against alcohol-induced injury in in vitro rat primary hepatocytes cell culture. This collaborate our previous observation of its hepatoprotective effect in a severe sustained 90-min hemorrhagic shock rat model.

Case Study: Organotypic human in vitro models of embryonic ...

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Morphogenetic fusion of tissues is a common event in embryonic development and disruption of fusion is associated with birth defects of the eye, heart, neural tube, phallus, palate, and other organ systems. Embryonic tissue fusion requires precise regulation of cell-cell and cell-matrix interactions that drive proliferation, differentiation, and morphogenesis. Chemical low-dose exposures can disrupt morphogenesis across space and time by interfering with key embryonic fusion events. The Morphogenetic Fusion Task uses computer and in vitro models to elucidate consequences of developmental exposures. The Morphogenetic Fusion Task integrates multiple approaches to model responses to chemicals that leaad to birth defects, including integrative mining on ToxCast DB, ToxRefDB, and chemical structures, advanced computer agent-based models, and human cell-based cultures that model disruption of cellular and molecular behaviors including mechanisms predicted from integrative data mining and agent-based models. The purpose of the poster is to indicate progress on the CSS 17.02 Virtual Tissue Models Morphogenesis Task 1 products for the Board of Scientific Counselors meeting on Nov 16-17.

A fraction enriched in rat hippocampal mossy fibre synaptosomes contains trophic activities.

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Taupin, P; Roisin, M P; Ben-Ari, Y; Barbin, G

1994-06-27

Subcellular fractions prepared from the rat hippocampus, were assessed for the presence of trophic activities. The cytosol of synaptosomal fractions induced mitotic reinitiation of confluent 3T3 fibroblasts. The synaptosomal fraction, enriched in mossy fibre terminals, contained the highest mitotic activity. The mitogenic activity was heat and trypsin sensitive, suggesting that polypeptides are involved. The cytosol of the mossy fibre synaptosomal fraction promoted neuritic outgrowth of PC 12 cells and embryonic hippocampal neurones in primary cultures. These results suggest that mossy fibres contain both mitogenic and neurotrophic activities. These factors could participate in mossy fibre sprouting that occur following brief seizures or experimental lesions.

Mitochondrial ribosomal protein S18-2 evokes chromosomal instability and transforms primary rat skin fibroblasts

KAUST Repository

Kashuba, Elena; Carbone, Ennio; Di Fabrizio, Enzo M.; Tirinato, Luca; Petruchek, Maria; Drummond, Catherine; Kovalevska, Larysa; Gurrapu, Sreeharsha; Mushtaq, Muhammad; Darekar, Suhas D.

2015-01-01

We have shown earlier that overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2) led to immortalization of primary rat embryonic fibroblasts. The derived cells expressed the embryonic stem cell markers, and cellular pathways

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells.

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Hunt, Geoffrey C; Singh, Purva; Schwarzbauer, Jean E

2012-09-10

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. Copyright © 2012 Elsevier Inc. All rights reserved.

How does blastomere removal affect embryonic development? : A time-lapse analysis

DEFF Research Database (Denmark)

Kirkegaard, Kirstine; Hindkjær, Johnny Juhl; Ingerslev, Hans Jakob

of the 6-10 cell embryo. It has been argued that blastomere removal does not affect embryonic development, but few studies have focussed on safety of the procedure. Recently, time-lapse studies on mice have suggested that blastomere removal affects embryonic development. The present study was conducted...... to evaluate the effect of blastomere biopsy on early human embryonic development using time-lapse analysis. Materials and methods: Couples undergoing IVF treatment or PGD were requested permission to include embryos in the project. The diagnosis healthy/diseased was made by analysis of a single blastomere....... For PGD 56 human embryos were biopsied 68 hours after fertilisation, the majority at the eight cell stage. As controls 43 non-biopsied embryos at the 6-8 cell stage were selected. All embryos were cultured until 5 days after fertilisation in a time-lapse incubator (EmbryoScope™). Key events such as time...

Embryonal carcinoma cell induction of miRNA and mRNA changes in co-cultured prostate stromal fibromuscular cells

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VÊNCIO, ENEIDA F.; PASCAL, LAURA E.; PAGE, LAURA S.; DENYER, GARETH; WANG, AMY J.; RUOHOLA-BAKER, HANNELE; ZHANG, SHILE; WANG, KAI; GALAS, DAVID J.; LIU, ALVIN Y.

2014-01-01

The prostate stromal mesenchyme controls organ-specific development. In cancer, the stromal compartment shows altered gene expression compared to non-cancer. The lineage relationship between cancer-associated stromal cells and normal tissue stromal cells is not known. Nor is the cause underlying the expression difference. Previously, the embryonal carcinoma (EC) cell line, NCCIT, was used by us to study the stromal induction property. In the current study, stromal cells from non-cancer (NP) and cancer (CP) were isolated from tissue specimens and co-cultured with NCCIT cells in a trans-well format to preclude heterotypic cell contact. After 3 days, the stromal cells were analyzed by gene arrays for microRNA (miRNA) and mRNA expression. In co-culture, NCCIT cells were found to alter the miRNA and mRNA expression of NP stromal cells to one like that of CP stromal cells. In contrast, NCCIT had no significant effect on the gene expression of CP stromal cells. We conclude that the gene expression changes in stromal cells can be induced by diffusible factors synthesized by EC cells, and suggest that cancer-associated stromal cells represent a more primitive or less differentiated stromal cell type. PMID:20945389

Effect of bionic electrical stimulation on the differentiation of embryonic stem cells into cardiomyocytes in the presence myocardial cells in vitro

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Li-na ZHENG

2011-08-01

Full Text Available Objective To investigate the effects of electrical stimulation on the differentiation of embryonic stem cells(ESCs into cardiomyocytes in the presence of myocardial cells in vitro.Methods ESCs and neonate rat cardiomyocytes were isolated and cultured.These cells of primary culture were divided into 5 groups according to whether or not electric stimulation was given and the presence of cardiomyocytes: control group,stimulation group,cardiomyocytes group,stimulation+ cardiomyocyte conditioned medium group,and stimulation+cardiomyocytes group.Expression of troponin T(cTnT in the differentiated cells from ESCs was examined by immunofluoresence on the 5th,7th and 14th day.Results In the group co-cultured with myocardial cell and electrical stimulation,the differentiating ratio of cardiomyocytes derived from ESCs and expressing cTnT was 40.00%±2.39%,and it was higher than that in control group(2.00%±1.60%,stimulation group(3.00%±2.00%,cardiomyocytes group(28.70%±4.06%,stimulation+cardiomyocyte conditioned medium group(17.10%±2.23%,P < 0.05.Conclusion Bionic electric stimulation promotes the differentiation of ESCs into cardiomyocyte in a microenvironment consisting of myocardial cells.

Development of buffalo (Bubalus bubalis embryonic stem cell lines from somatic cell nuclear transferred blastocysts

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Syed Mohmad Shah

2015-11-01

Full Text Available We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

Survival and Functionality of hESC-Derived Retinal Pigment Epithelium Cells Cultured as a Monolayer on Polymer Substrates Transplanted in RCS Rats.

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Thomas, Biju B; Zhu, Danhong; Zhang, Li; Thomas, Padmaja B; Hu, Yuntao; Nazari, Hossein; Stefanini, Francisco; Falabella, Paulo; Clegg, Dennis O; Hinton, David R; Humayun, Mark S

2016-05-01

To determine the safety, survival, and functionality of human embryonic stem cell-derived RPE (hESC-RPE) cells seeded on a polymeric substrate (rCPCB-RPE1 implant) and implanted into the subretinal (SR) space of Royal College of Surgeons (RCS) rats. Monolayers of hESC-RPE cells cultured on parylene membrane were transplanted into the SR space of 4-week-old RCS rats. Group 1 (n = 46) received vitronectin-coated parylene membrane without cells (rMSPM+VN), group 2 (n = 59) received rCPCB-RPE1 implants, and group 3 (n = 13) served as the control group. Animals that are selected based on optical coherence tomography screening were subjected to visual function assays using optokinetic (OKN) testing and superior colliculus (SC) electrophysiology. At approximately 25 weeks of age (21 weeks after surgery), the eyes were examined histologically for cell survival, phagocytosis, and local toxicity. Eighty-seven percent of the rCPCB-RPE1-implanted animals showed hESC-RPE survivability. Significant numbers of outer nuclear layer cells were rescued in both group 1 (rMSPM+VN) and group 2 (rCPCB-RPE1) animals. A significantly higher ratio of rod photoreceptor cells to cone photoreceptor cells was found in the rCPCB-RPE1-implanted group. Animals with rCPCB-RPE1 implant showed hESC-RPE cells containing rhodopsin-positive particles in immunohistochemistry, suggesting phagocytic function. Superior colliculus mapping data demonstrated that a significantly higher number of SC sites responded to light stimulus at a lower luminance threshold level in the rCPCB-RPE1-implanted group. Optokinetic data suggested both implantation groups showed improved visual acuity. These results demonstrate the safety, survival, and functionality of the hESC-RPE monolayer transplantation in an RPE dysfunction rat model.

Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

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Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

2017-10-01

Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Effect of Co-Culturing of Mice Liver Cells and Embryonic Carcinomatous Stem Cells on the Rate of Differentiation to Hematopoietic Cells

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AA Pourfatollah

2005-10-01

Full Text Available Introduction: Considering the importance of co-culture in differentiation of embryonic stem cells, the aim of this study was evaluation of the effect of co-culturing fetal liver stroma cells with P19 cells on the line of differentiation. Materials and Methods: For this purpose, P19 cells were cultured directly in semisolid medium. These cells proliferated and primarily differentiated to colonies know as embryoid bodies (EBs after 8-12 days. The Ebs cells were trypsinized and dissociated to single or double cells. Then these cells were co-cultured on the mouse fetal liver feeder layer in the absence of exogenous factors. After 14-18 days, the colonies were studied morphologically by benzidine and giemsa staining and also counted under invert microscope. Results: The percentages of benzidine positive (or erythroid and negative colonies were 94% and 6% respectively and also the cells of colonies were studied by Giemsa staining. Results showed that they were myeloid or lymphoid type cells. Thus, the results show that in the presence of mouse fetal liver feeder layer, the number of erythroid colonies was increased. Conclusions: Therefore, this technique may be effective for differentiation of stem cells from different sources into hematopoietic cells and can be used in future for human cell therapy.

Storing live embryonic and adult human cartilage grafts for transplantation using a joint simulating device.

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Cohen, I; Robinson, D; Cohen, N; Nevo, Z

2000-11-01

Cartilage transplantation as a means to replace damaged articular surfaces is of interest. A major obstacle is the long-term preservation of cartilage grafts. The commonly used technique of freezing the grafts inevitably leads to cellular death. The current study compares the technique to an innovative approach using a pulsed-pressure perfusion system termed a joint simulating device (JSD), intended to simulate intra-articular mechanical forces. Human articular cartilage explants were harvested from both embryonic epiphyseal tissue and femoral heads of elderly women (over 70 years of age) undergoing a partial joint replacement (hemi-arthroplasty) and were divided in two groups: half of the samples were incubated in the JSD while the remaining half were grown in static culture within tissue culture plates. After 10 days all samples were evaluated for: (a) cell vitality as assessed by image analysis and XTT assay; (b) biosynthetic activity as expressed by radioactive sulfate incorporation into glycosaminoglycans (GAG's); and (c) proteoglycan content as assessed by alcian blue staining intensity. A 10-fold increase in sulfate incorporation in samples held in the JSD compared to the static culture group was observed in embryonic cartilage. In adult cartilage culture in the JSD elevated sulfate incorporation by threefold as compared to static culture. Central necrosis was observed in specimens grown in the static culture plates, while it did not occur in the samples held in the JSD. Cell vitality as assessed by XTT assay was significantly better in the JSD group as compared to static culture. The difference was more pronounced in the embryonic specimens as compared to adult cartilage. The specimens cultured within the JSD retained proteoglycans significantly better than those cultured in static culture. Maintenance of cartilage specimens in a JSD was highly effective in keeping the vitality of cartilage explants in vitro over a 10-day period. A possible future

Alterations in the developing testis transcriptome following embryonic vinclozolin exposure.

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Clement, Tracy M; Savenkova, Marina I; Settles, Matthew; Anway, Matthew D; Skinner, Michael K

2010-11-01

The current study investigates the direct effects of in utero vinclozolin exposure on the developing F1 generation rat testis transcriptome. Previous studies have demonstrated that exposure to vinclozolin during embryonic gonadal sex determination induces epigenetic modifications of the germ line and transgenerational adult onset disease states. Microarray analyses were performed to compare control and vinclozolin treated testis transcriptomes at embryonic days 13, 14 and 16. A total of 576 differentially expressed genes were identified and the major cellular functions and pathways associated with these altered transcripts were examined. The sets of regulated genes at the different development periods were found to be transiently altered and distinct. Categorization by major known functions of altered genes was performed. Specific cellular process and pathway analyses suggest the involvement of Wnt and calcium signaling, vascular development and epigenetic mechanisms as potential mediators of the direct F1 generation actions of vinclozolin. Copyright © 2010 Elsevier Inc. All rights reserved.

Changes in expression of a functional Gi protein in cultured rat heart cells

International Nuclear Information System (INIS)

Allen, I.S.; Gaa, S.T.; Rogers, T.B.

1988-01-01

The muscarinic cholinergic agonist, carbachol, and pertussis toxin were used to examine the functional status of the guanine nucleotide-binding protein that inhibits adenylate cyclase (G i ) in cultured neonatal rat heart myocytes. The isoproterenol stimulation of adenylate cyclase activity in myocyte membranes and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in intact cells (4 days in culture) were insensitive to carbachol. However, in cells cultured for 11 days, carbachol inhibited isoproterenol-stimulated cAMP accumulation by 30%. Angiotensin II (ANG II) was also found to inhibit isoproterenol-stimulated cAMP accumulation in day 11 cells in a dose-dependent manner. Pertussis toxin treatment reversed the inhibitory effects of both ANG II and carbachol, suggesting a role for G i in the process. Carbachol binding to membranes from day 4 cells was relatively insensitive to guanine nucleotides when compared with binding to membranes from day 11 or adult cells. Furthermore, pertussis toxin-mediated 32 P incorporation into a 39- to 41-kDa substrate in day 11 membranes was increased 3.2-fold over that measured in day 4 membranes. These findings support the view that, although G i is expressed, it is nonfunctional in 4-day-old cultured neonatal rat heart myocytes and acquisition of functional G i is dependent on culture conditions. Furthermore, the ANG II receptor can couple to G i in heart

[Expression of embryonic markers in pterygium derived mesenchymal cells].

Science.gov (United States)

Pascual, G; Montes, M A; Pérez-Rico, C; Pérez-Kohler, B; Bellón, J M; Buján, J

2010-12-01

Destruction of the limbal epithelium barrier is the most important mechanism of pterygium formation (conjunctiva proliferation, encroaching onto the cornea). It is thought to arise from activated and proliferating limbal epithelial stem cells. The objective of this study is to evaluate the presence of undifferentiated mesenchymal cells (stem cells) in cultured cells extracted from human pterygium. Cells from 6 human pterygium were isolated by explantation and placed in cultures with amniomax medium. Once the monolayer was reached the cells were seeded onto 24 well microplates. The cells were studied in the second sub-culture. The immunohistochemical expression of different embryonic stem cell markers, OCT3/4 and CD9, was analysed. The differentiated phenotypes were characterised with the monoclonal antibodies anti-CD31, α-actin and vimentin. All the cell populations obtained from pterygium showed vimentin expression. Less than 1% of the cells were positive for CD31 and α-actin markers. The majority of the cell population was positive for OCT3/4 and CD9. The cell population obtained from pterygium expressed mesenchymal cell phenotype and embryonic markers, such us OCT3/4 and CD9. This undifferentiated population could be involved in the large recurrence rate of this type of tissue after surgery. Copyright © 2010 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

Induction of peroxisomal beta-oxidation by a microbial catabolite of cholic acid in rat liver and cultured rat hepatocytes.

Science.gov (United States)

Nishimaki-Mogami, T; Takahashi, A; Toyoda, K; Hayashi, Y

1993-01-01

The capability of (4R)-4-(2,3,4,6,6a beta,7,8,9,9a alpha,9b beta-decahydro-6a beta-methyl-3-oxo-1H-cyclopental[f]quinolin-7 beta-yl)valeric acid (DCQVA), a catabolite of cholic acid produced by enterobacteria, to induce peroxisome proliferation in vivo and in vitro was studied. Rats given 0.3% DCQVA in the diet for 2 weeks showed marked increases in peroxisomal beta-oxidation, mitochondrial 2,4-dienoyl-CoA reductase and microsomal laurate omega-oxidation activities in the liver compared with control rats given the diet without DCQVA. Cultured rat hepatocytes treated with DCQVA for 72 h also exhibited greatly enhanced beta-oxidation activity. The increased activity was concentration-dependent and the effective concentrations were comparable with those of clofibric acid that produced the same degree of induction in the assay. The results demonstrate that DCQVA is a potent peroxisome proliferator that occurs naturally in rat intestine. PMID:8216219

Tissue culture of osteogenic sarcoma in rats, induced by radioactive phosphorus P-32 and the effect of the anti-cancerous agents on these tumor cells under tissue culture

International Nuclear Information System (INIS)

Osaka, Shunzo

1976-01-01

Small pieces of osteogenic sarcoma, induced into albino rats of the C.F. Wistar strain by injection of radioactive phosphorus 32 P, were cultured in mixtures of Eagle's minimum essential medium and 20% calf serum. The tumor cells cultured in this way were transplanted into the subcutaneous tissue or the intraabdominal cavity to healthy albino rats. The effect of the anticancerous agents was evaluated by the decrease of nucleic acid composition in these cultured tumor cells. As anti-cancerous agents, cyclophosphamide (CPA), mitomycin C(MMC), and 5-fluorouracil(5-FU) were put into contact with the tumor cells in cultures for two hours under the following dilutions: CPA; 10 -6 , 10 -5 , 10 -4 g/ml. MMC; 2 x 10 -8 , 2 x 10 -7 , 2 x 10 -6 g/ml. 5-FU; 2 x 10 -6 , 2 x 10 -5 , 2 x 10 -4 g/ml. The results are as follows: Three of the seven osteogenic sarcomas in rats were successfully cultured, one of them through more than eighteen generations. After about five hundred thousand cultured cells had been transplanted into the subcutaneous tissues or abdominal cavities of rats, tumors grew in all of them. The histological findings of the tumors in the second generation were quite similar to those of the original tumor. The same process was repeated three times and the tumor showed histogical findings similar to those of the original ones. The capability of nucleic acid synthesis in these cells was decreased at twenty fours after CPA contact and at forty eight hours after MMC. (J.P.N.)

Do embryonic polar bodies commit suicide?

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Fabian, Dušan; Čikoš, Štefan; Rehák, Pavol; Koppel, Juraj

2014-02-01

The extrusion and elimination of unnecessary gametic/embryonic material is one of the key events that determines the success of further development in all living organisms. Oocytes produce the first polar body to fulfill the maturation process just before ovulation, and release the second polar body immediately after fertilization. The aim of this study was to compile a physiological overview of elimination of polar bodies during early preimplantation development in mice. Our results show that three-quarters of the first polar bodies were lost even at the zygotic stage; the 4-cell stage embryos contained only one (second) polar body, and the elimination of second polar bodies proceeded continuously during later development. Both first and second polar bodies showed several typical features of apoptosis: phosphatidylserine redistribution (observed for the first time in the first polar body), specific DNA degradation, condensed nuclear morphology, and inability to exclude cationic dye from the nucleus during the terminal stage of the apoptotic process. Caspase-3 activity was recorded only in the second polar body. From the morphological point of view, mouse polar bodies acted very similarly to damaged embryonic cells which have lost contact with their neighboring blastomeres. In conclusion, polar bodies possess all the molecular equipment necessary for triggering and executing an active suicide process. Furthermore, similarly as in dying embryonic cells, stressing external conditions (culture in vitro) might accelerate and increase the incidence of apoptotic elimination of the polar bodies in embryos.

Growth factor expression pattern of homologous feeder layer for culturing buffalo embryonic stem cell-like cells.

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Sharma, Ruchi; George, Aman; Kamble, Nitin M; Chauhan, Manmohan S; Singla, Suresh; Manik, Radhey S; Palta, Prabhat

2012-01-01

The present study examined the expression profile of buffalo fetal fibroblasts (BFF) used as a feeder layer for embryonic stem (ES) cell-like cells. The expression of important growth factors was detected in cells at different passages. Mitomycin-C inactivation increased relative expression levels of ACTIVIN-A, TGF-β1, BMP-4 and GREMLIN but not of fibroblast growth factor-2 (FGF-2). The expression level of ACTIVIN-A, transforming growth factor-β1 (TGF-β1), bone morphogenetic protein-4 (BMP-4) and FGF-2 was similar in buffalo fetal fibroblast (BFF) cultured in stem cell medium (SCM), SCM+1000IU mL(-1) leukemia inhibitory factor (LIF), SCM+5 ngmL(-1) FGF-2 or SCM+LIF+FGF-2 for 24 h whereas GREMLIN expression was higher in FGF-2-supplemented groups. In spent medium, the concentration of ACTIVIN-A was higher in FGF-2-supplemented groups whereas that of TGF-β1 was similar in SCM and LIF+FGF-2, which was higher than when either LIF or FGF-2 was used alone. Following culture of ES cell-like cells on a feeder layer for 24 h, the TGF-β1 concentration was higher with LIF+FGF-2 than with LIF or FGF-2 alone which, in turn, was higher than that in SCM. In the LIF+FGF-2 group, the concentration of TGF-β1 was lower and that of ACTIVIN-A was higher in spent medium at 24 h than at 48 h of culture. These results suggest that BFF produce signalling molecules that may help in self-renewal of buffalo ES cell-like cells.

A new pH-sensitive rectifying potassium channel in mitochondria from the embryonic rat hippocampus.

Science.gov (United States)

Kajma, Anna; Szewczyk, Adam

2012-10-01

Patch-clamp single-channel studies on mitochondria isolated from embryonic rat hippocampus revealed the presence of two different potassium ion channels: a large-conductance (288±4pS) calcium-activated potassium channel and second potassium channel with outwardly rectifying activity under symmetric conditions (150/150mM KCl). At positive voltages, this channel displayed a conductance of 67.84pS and a strong voltage dependence at holding potentials from -80mV to +80mV. The open probability was higher at positive than at negative voltages. Patch-clamp studies at the mitoplast-attached mode showed that the channel was not sensitive to activators and inhibitors of mitochondrial potassium channels but was regulated by pH. Moreover, we demonstrated that the channel activity was not affected by the application of lidocaine, an inhibitor of two-pore domain potassium channels, or by tertiapin, an inhibitor of inwardly rectifying potassium channels. In summary, based on the single-channel recordings, we characterised for the first time mitochondrial pH-sensitive ion channel that is selective for cations, permeable to potassium ions, displays voltage sensitivity and does not correspond to any previously described potassium ion channels in the inner mitochondrial membrane. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). Copyright © 2012 Elsevier B.V. All rights reserved.

Evaluation of castor oil-based polyurethane membranes in rat bone-marrow cell culture.

Science.gov (United States)

Cerejo, Sofia de Amorim; Rahal, Sheila Canevese; Lima Neto, João Ferreira de; Voorwald, Fabiana Azevedo; Alvarenga, Fernanda da Cruz Landim e

2011-10-01

To evaluate three methods to isolate rats MSCs and to analyze the potential of a castor oil polyurethane base membrane as a scaffold for MSCs. Four male Wistar rats, aged 20-30 days were used. Bone marrow aspirates from femur and tibia were harvested using DMEM high glucose and heparin. The cell culture was performed in three different ways: direct culture and two types of density gradients. After 15 days, was made the 1st passage and analyzed cell viability with markers Hoerscht 33342 and propidium iodide. The MSCs were characterized by surface markers with the aid of flow cytometry. After this, three types of castor oil polyurethane membranes associated with the MSCs were kept on the 6-well plate for 5 days and were analyzed by optical microscopy to confirm cell aggregation and growth. Separation procedures 1 and 2 allowed adequate isolation of MSCs and favored cell growth with the passage being carried out at 70% confluence after 15 days in culture. The cells could not be isolated using procedure 3. When the 3 castor oil polyurethane membrane types were compared it was possible to observe that the growth of MSCs was around 80% in membrane type 3, 20% in type 2, and 10% in type 1. Both Ficoll-Hypaque densities allow isolation of rat MSCs, and especially castor oil-based membrane type 3 may be used as a scaffold for MSCs.

Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

DEFF Research Database (Denmark)

Li, Dong; Secher, Jan Ole Bertelsen; Juhl, Morten

2017-01-01

Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells...... are the only cells capable of being reprogrammed from a heterogeneous population of fibroblasts. Similarly, there is little research to suggest such cells may exist in embryonic tissues or other species. To address if such a cell population exists in pigs, we investigated porcine embryonic fibroblast...... populations (pEFs) and identified heterogeneous expression of several key cell surface markers. Strikingly, we discovered a small population of stage-specific embryonic antigen 1 positive cells (SSEA-1+) in Danish Landrace and Göttingen minipig pEFs, which were absent in the Yucatan pEFs. Furthermore...

Immunofluorescent histological studies of the role of fibronectin in the expression of the associative preferences of embryonic tissues.

Science.gov (United States)

Armstrong, P B; Armstrong, M T

1981-08-01

The identity of the chemical factors controlling the spreading behaviour of sheets of cells was examined in organ culture. When aggregates of two dissimilar tissues are apposed in organ culture, one tissue spreads reproducibly over the surface of the second. The present study employed indirect immunofluorescent localization techniques to evaluate the hypothesis that the spreading behaviour of chick embryonic heart tissue in culture is dominated by the presence or absence of the cell-surface and extracellular matrix protein fibronectin in the surface layers of the aggregates. Specifically, the hypothesis proposes that aggregates that display surface fibronectin earlier after culturing and/or in higher quantities segregate internally to aggregates that are slower to develop a surface layer of fibronectin or in which this layer contains reduced amounts of fibronectin. The hypothesis has been supported for 3 categories of behaviour of chick embryo heart tissue: (1) myocyte aggregates spread over myocyte aggregates containing a 20% admixture of heart fibroblasts, which in turn spread over heart fibroblast aggregates; (2) 5-day embryonic ventricle-tissue fragments maintained in culture for 0.5 days spread over ventricle fragments cultured for 2.5 days; and (3) 2-day embryonic ventricle spreads over 5-day ventricle. In all these situations, the aggregate type that segregates to an internal position displays more fibronectin at its surface than aggregate types that spread to occupy an external position. Evidence is presented that the fibronectin in heart tissue aggregates is elaborated by heart fibroblasts.

Culture conditions have an impact on the maturation of traceable, transplantable mouse embryonic stem cell-derived otic progenitor cells.

Science.gov (United States)

Abboud, Nesrine; Fontbonne, Arnaud; Watabe, Isabelle; Tonetto, Alain; Brezun, Jean Michel; Feron, François; Zine, Azel

2017-09-01

The generation of replacement inner ear hair cells (HCs) remains a challenge and stem cell therapy holds the potential for developing therapeutic solutions to hearing and balance disorders. Recent developments have made significant strides in producing mouse otic progenitors using cell culture techniques to initiate HC differentiation. However, no consensus has been reached as to efficiency and therefore current methods remain unsatisfactory. In order to address these issues, we compare the generation of otic and HC progenitors from embryonic stem (ES) cells in two cell culture systems: suspension vs. adherent conditions. In the present study, an ES cell line derived from an Atoh1-green fluorescent protein (GFP) transgenic mouse was used to track the generation of otic progenitors, initial HCs and to compare these two differentiation systems. We used a two-step short-term differentiation method involving an induction period of 5 days during which ES cells were cultured in the presence of Wnt/transforming growth factor TGF-β inhibitors and insulin-like growth factor IGF-1 to suppress mesoderm and reinforce presumptive ectoderm and otic lineages. The generated embryoid bodies were then differentiated in medium containing basic fibroblast growth factor (bFGF) for an additional 5 days using either suspension or adherent culture methods. Upon completion of differentiation, quantitative polymerase chain reaction analysis and immunostaining monitored the expression of otic/HC progenitor lineage markers. The results indicate that cells differentiated in suspension cultures produced cells expressing otic progenitor/HC markers at a higher efficiency compared with the production of these cell types within adherent cultures. Furthermore, we demonstrated that a fraction of these cells can incorporate into ototoxin-injured mouse postnatal cochlea explants and express MYO7A after transplantation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons

Generation of primary cultures of bovine brain endothelial cells and setup of cocultures with rat astrocytes

DEFF Research Database (Denmark)

Helms, Hans C; Brodin, Birger

2014-01-01

-brain barrier. The present protocol describes the setup of an in vitro coculture model based on primary cultures of endothelial cells from bovine brain microvessels and primary cultures of rat astrocytes. The model displays a high electrical tightness and expresses blood-brain barrier marker proteins....

optimization of protocol for m apical meristem of embryonic axes

African Journals Online (AJOL)

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The hypocotyl segments, prima were removed and embryonic axes were placed in culture bott nd Skoog basal + B5 vitamins (MSB5) fortified with 22.2µM, 26.6µM opurine (BAP) to induce multiple shoots. Elongated shoots we tion media consisting of MSB5 supplemented with low concentrati one free media promoted direct ...

Ouabain binding to cultured vascular smooth muscle cells of the spontaneously hypertensive rat

International Nuclear Information System (INIS)

Hopp, L.; Khalil, F.; Tamura, H.; Kino, M.; Searle, B.M.; Tokushige, A.; Aviv, A.

1986-01-01

The binding of ouabain and K + to the Na + pump were analyzed in serially passed cultured vascular smooth muscle cells (VSMCs) originating from spontaneously hypertensive (SH) Wistar-Kyoto (WKY), and American Wistar (W) rats. The techniques have utilized analyses of displacement of [ 3 H]ouabain by both unlabeled ouabain and K + from specific binding sites on the VSMCs. The authors have found that 1) each of the VSMC preparations from the three rat strains appeared to demonstrate one population of specific ouabain receptors (Na + pumps); 2) the number of Na + pump units of both the SH and WKY rats was significantly lower than the number of Na + pump units of W rat VSMCs; 3) the equilibrium dissociation constant values (μM) for ouabain in VSMCs of SH and WKY rats were similar but were significantly higher than that of VSMCs derived from W rats; and 4) among the VSMCs originating from the three rat strains, the apparent equilibrium dissociation constant value for K + (mM) was the lowest in those of the SH rat compared with VSMCs of the WKY rat and W rat. Previous studies have demonstrated increased passive Na + and K + transport rate constants of SH rat VSMCs compared with either W or WKY rat cells. These findings suggest the possibility of higher permeabilities of the SH cells. They propose that the combined effect of a low number of Na + pump units with higher permeabilities to Na + and K + predisposes VSMCs of the SH rat to disturbances in their cellular ionic regulation. These genetic defects, if they occur in vivo, may lead to an increase in the vascular tone

Control of the heart rate of rat embryos during the organogenic period

Directory of Open Access Journals (Sweden)

Ritchie HE

2016-11-01

Full Text Available Helen E Ritchie,1 Carolina Ragnerstam,2 Elin Gustafsson,2 Johanna M Jonsson,2 William S Webster2 1Discipline of Biomedical Science, Sydney Medical School, University of Sydney, Lidcombe, 2Department of Anatomy and Histology, Sydney Medical School, University of Sydney, Sydney, NSW, Australia Abstract: The aim of this study was to gain insight into whether the first trimester embryo could control its own heart rate (HR in response to hypoxia. The gestational day 13 rat embryo is a good model for the human embryo at 5–6 weeks gestation, as the heart is comparable in development and, like the human embryo, has no functional autonomic nerve supply at this stage. Utilizing a whole-embryo culture technique, we examined the effects of different pharmacological agents on HR under normoxic (95% oxygen and hypoxic (20% oxygen conditions. Oxygen concentrations ≤60% caused a concentration-dependent decrease in HR from normal levels of ~210 bpm. An adenosine agonist, AMP-activated protein kinase (AMPK activator and KATP channel opener all caused bradycardia in normoxic conditions; however, putative antagonists for these systems failed to prevent or ameliorate hypoxia-induced bradycardia. This suggests that the activation of one or more of these systems is not the primary cause of the observed hypoxia-induced bradycardia. Inhibition of oxidative phosphorylation also decreased HR in normoxic conditions, highlighting the importance of ATP levels. The β-blocker metoprolol caused a concentration-dependent reduction in HR supporting reports that β1-adrenergic receptors are present in the early rat embryonic heart. The cAMP inducer colforsin induced a positive chronotropic effect in both normoxic and hypoxic conditions. Overall, the embryonic HR at this stage of development is responsive to the level of oxygenation, probably as a consequence of its influence on ATP production. Keywords: embryonic heart rate, embryo, bradycardia, in vitro, ATP, hypoxia

Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery

DEFF Research Database (Denmark)

Kiprilov, Enko N; Awan, Aashir; Desprat, Romain

2008-01-01

Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human hESC d...

Feeding Frequency Affects Cultured Rat Pituitary Cells in Low Gravity

Science.gov (United States)

Hymer, W. C.; Grindeland, R. E.; Salada, T.; Cenci, R.; Krishnan, K.; Mukai, C.; Nagaoka, S.

1996-01-01

In this report, we describe the results of a rat pituitary cell culture experiment done on STS-65 in which the effect of cell feeding on the release of the six anterior pituitary hormones was studied. We found complex microgravity related interactions between the frequency of cell feeding and the quantity and quality (i.e. biological activity) of some of the six hormones released in flight. Analyses of growth hormone (GH) released from cells into culture media on different mission days using gel filtration and ion exchange chromatography yielded qualitatively similar results between ground and flight samples. Lack of cell feeding resulted in extensive cell clumping in flight (but not ground) cultures. Vigorous fibroblast growth occurred in both ground and flight cultures fed 4 times. These results are interpreted within the context of autocrine and or paracrine feedback interactions. Finally the payload specialist successfully prepared a fresh trypsin solution in microgravity, detached the cells from their surface and reinserted them back into the culture chamber. These cells reattached and continued to release hormone in microgravity. In summary, this experiment shows that pituitary cells are microgravity sensitive and that coupled operations routinely associated with laboratory cel1 culture can also be accomplished in low gravity.

Lack of direct mitogenic activity of dichloroacetate and trichloroacetate in cultured rat hepatocytes

International Nuclear Information System (INIS)

Walgren, Jennie L.; Kurtz, David T.; McMillan, JoEllyn M.

2005-01-01

Dichloroacetate (DCA) and trichloroacetate (TCA) are hepatocarcinogenic metabolites of the common groundwater contaminant, 1,1,2-trichloroethylene. DCA and TCA have been shown to induce hepatocyte proliferation in vivo, but it is not known if this response is the result of direct mitogenic activity or whether cell replication occurs indirectly in response to tissue injury or inflammation. In this study we used primary cultures of rat hepatocytes, a species susceptible to DCA- but not TCA-induced hepatocarcinogenesis, to determine whether DCA and TCA are direct hepatocyte mitogens. Rat hepatocytes, cultured in growth factor-free medium, were treated with 0.01-1.0 mM DCA or TCA for 10-40 h; cell replication was then assessed by measuring incorporation of 3 H-thymidine into DNA and by cell counts. DCA or TCA treatment did not alter 3 H-thymidine incorporation in the cultured hepatocytes. Although an increase in cell number was not observed, DCA treatment significantly abrogated the normal background cell loss, suggesting an ability to inhibit apoptotic cell death in primary hepatocyte cultures. Furthermore, treatment with DCA synergistically enhanced the mitogenic response to epidermal growth factor. The data indicate that DCA and TCA are not direct mitogens in hepatocyte cultures, which is of interest in view of their ability to stimulate hepatocyte replication in vivo. Nevertheless, the synergistic enhancement of epidermal growth factor-induced hepatocyte replication by DCA is of particular interest and warrants further study

Neurochemical and toxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenylpyridine to rat serotonin neurons in dissociated cell cultures

International Nuclear Information System (INIS)

Friedman, L.K.; Mytilineou, C.

1990-01-01

Dissociated cell cultures from the pontine area of embryonic rat brain were used to study the sensitivity of serotonin (5-hydroxy-tryptamine (5-HT)) neurons to the neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine (MPP+). Treatment with MPTP (up to 100 microM) for 7 days did not cause degeneration of 5-HT neurons. A 50% inhibition of [3H]5-HT uptake caused by 100 microM MPTP was a direct effect on the 5-HT uptake carrier, reversed by washing for 7 days. Incubation of cultures with MPTP increased the intraneuronal levels of 5-HT and reduced the levels of 5-hydroxyindoleacetic acid, suggesting a reduction in 5-HT metabolism. MPTP reduced monoamine oxidase activity in the cultures, which probably led to the reduction in 5-HT metabolism. Exposure to MPP+ (0.5-10 microM) for 4 to 7 days decreased [3H]5-HT uptake and induced loss of neurons stained with antibodies against 5-HT. Comparison between 5-HT and dopamine (DA) neurons indicated a differential sensitivity to MPP+ toxicity with DA neurons being more susceptible. Analysis of the competition of MPP+ with the natural substrates for uptake sites of 5-HT and DA neurons demonstrated higher affinity of MPP+ for DA compared to 5-HT neurons. The lower affinity of MPP+ for 5-HT neurons could be responsible for the accumulation of lower MPP+ levels observed in pontine cultures and explain the resistance of 5-HT neurons to this toxin

Embryonic skeleton development and neonatal learning and memory ability of rats anesthetized with pentobarbital sodium: Differences of administration occasion and time

Institute of Scientific and Technical Information of China (English)

Changling Peng; Yuhua Zhu; Ankang Hu; Xiaorong Zhu

2006-01-01

BACKGROUND : Generally speaking, anesthesia is often used in gravid body and it has been already proved that many kind of medicine can result in malformation.OBJECTIVE: To explore embryonic skeleton development and neonatal learning and memory of rats anesthetized with pentobarbital sodium in gravid rats.DESIGN: A randomized control trial.SETTING: Laboratory Animal Center of Xuzhou Medical College.MATERIALS: A total of 80 adult female SD rats, of clean grade and weighing 220-240 g, were selected in this study. The main reagents were detailed as follows: pentobarbital sodium (Shanghai Xingzhi Chemical Plant,batch number: 921019); MG-2 maze test apparatus (Zhangjiagang Biomedical Instrument Factory);somatotype microscope (Beijing Taike Instrument Co., Ltd.).METHODS: ① A total of 160 SD rats of half males and females were selected in this study. All rats were copulated. The day that the plug was checked out in the vagina next day was looked as the first day of pregnancy. Gravid rats were divided randomly into four groups, including early anesthesia group, second anesthesia group, late anesthesia group and control group with 20 in each group. Rats in the early anesthesia group were injected with 25 mg/kg soluble pentobarbitone on the 7th day of pregnancy for once; rats in the second anesthesia group were anesthetized with 25 mg/kg soluble pentobarbitone on the 7th and the 14th days of pregnancy for once; rats in the late anesthesia group were anesthetized with 25 mg/kg soluble pentobarbitone on the 14th day of pregnancy for once; rats in the control group did not treat with anything. The time of anesthetizing was controlled in 3 to 4 hours and ether was absorbed while the time was not enough. ②Half of each group was sacrificed on day 20th of pregnancy and the fetus was taken out to be stained with alizarin red S. After stained, the fetal skeleton was examined. The learning and memorizing of one-month rats that were given birth by the rest gravid rats were tested

Rat glomerular epithelial cells in culture. Parietal or visceral epithelial origin

International Nuclear Information System (INIS)

Norgaard, J.O.

1987-01-01

Isolated glomeruli from rats were explanted under standard culture conditions and outgrowths were studied by light and electron microscopy in order to identify the cells. Rat glomerular samples contained 20 to 30% structurally well-preserved encapsulated glomeruli which had a large rate of attachment to the substrate and very constantly gave rise to cellular outgrowth. In order to label cells from which outgrowth originated the glomerular incorporation of [ 3 H]thymidine was studied in the preattachment phase. By light and electron microscope autoradiograph it was demonstrated that label was located only over visceral and parietal epithelial cells during the first 3 days of culture. Incorporation of [ 3 H]thymidine was seen in mesangial cells after 5 days, i.e., after the glomeruli had attached to the culture vessels and the initial outgrowth had appeared. Consequently the first cells to grow out were of epithelial origin. Glomeruli were then incubated with [ 3 H]thymidine for the first 2 1/2 days of culture in order to label the epithelial cells, then were allowed to attach to the substrate and induce cell outgrowth. By light microscope autoradiography performed with the outgrowths in situ two types of cells with labeled nuclei were seen: (a) a small, polyhedral ciliated cell which grew in colonies where the cells were joined by junctional complexes (type I), and (b) a second very large, often multinucleated cell (type II). Based on the structural resemblance with their counterparts in situ and on comparisons with positively identified visceral epithelial cells in outgrowths from other species it is suggested that type I cells are derived from the parietal epithelium of Bowman's capsule and type II cells from the visceral epithelium

Ionic currents and charge movements in organ-cultured rat skeletal muscle.

Science.gov (United States)

Hollingworth, S; Marshall, M W; Robson, E

1984-12-01

The middle of the fibre voltage-clamp technique was used to measure ionic currents and non-linear charge movements in intact, organ-cultured (in vitro denervated) mammalian fast-twitch (rat extensor digitorum longus) muscle fibres. Muscle fibres organ cultured for 4 days can be used as electrophysiological and morphological models for muscles in vivo denervated for the same length of time. Sodium currents in organ-cultured muscle fibres are similar to innervated fibres except that in the temperature range 0-20 degrees C (a) in the steady state, the voltage distribution of inactivation in cultured fibres is shifted negatively some 20 mV; (b) at the same temperature and membrane potential, the time constant of inactivation in cultured fibres is about twice that of innervated fibres. Potassium currents in innervated and cultured fibres at 15 degrees C can be fitted with the Hodgkin-Huxley n variable raised to the second power. Despite the large range we would estimate that the maximum value of the steady-state potassium conductance of cultured fibres is about one-half that of innervated fibres. The estimated maximum amount of charge moved in cultured fibre is about one-third that in innervated fibres. Compared to innervated fibres, culturing doubles the kinetics of the decay phase of charge movement. The possibility of a negative shift of the voltage distribution of charge movements in cultured fibres is discussed.

Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics

Science.gov (United States)

Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultur...

Essential alterations of heparan sulfate during the differentiation of embryonic stem cells to Sox1-enhanced green fluorescent protein-expressing neural progenitor cells.

NARCIS (Netherlands)

Johnson, C.E.; Crawford, B.E.; Stavridis, M.; Dam, G.B. ten; Wat, A.L.; Rushton, G.; Ward, C.M.; Wilson, V.; Kuppevelt, A.H.M.S.M. van; Esko, J.D.; Smith, A.; Gallagher, J.T.; Merry, C.L.

2007-01-01

Embryonic stem (ES) cells can be cultured in conditions that either maintain pluripotency or allow differentiation to the three embryonic germ layers. Heparan sulfate (HS), a highly polymorphic glycosaminoglycan, is a critical cell surface coreceptor in embryogenesis, and in this paper we describe

Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos.

Science.gov (United States)

Chang, Tien-Cheng; Liu, Ya-Guang; Eddy, Carlton A; Jacoby, Ethan S; Binkley, Peter A; Brzyski, Robert G; Schenken, Robert S

2011-06-01

The development of nonhuman primate (NHP) embryonic stem cell (ESC) models holds great promise for cell-mediated treatment of debilitating diseases and to address numerous unanswered questions regarding the therapeutic efficacy of ESCs while supplanting ethical considerations involved with human studies. Here we report successful establishment and characterization of 3 novel baboon (Papio cynocephalus) ESC lines from the inner cell mass of intracytoplasmic sperm injection-derived blastocysts. Embryos were cultured in an improved baboon embryo in vitro culture protocol. The inner cell mass of blastocyst was laser-dissected and plated on mouse embryonic fibroblast feeder cell monolayer in the NHP ESC culture medium. Three cell lines with characteristic ESC morphology have been cultured through an extended period (>14 months), with 2 male cell lines (UT-1 and -2) and 1 female cell line (UT-3) displaying normal baboon karyotypes. Reverse transcription-polymerase chain reaction analysis confirmed that all 3 lines express primate ESC pluripotency markers, including OCT-4, NANOG, SOX-2, TERT, TDGF, LEFTYA, and REX-1. All 3 lines demonstrated positive immunocytochemical staining for OCT-4, stage-specific embryonic antigen-3, stage-specific embryonic antigen-4, TRA-1-60, and TRA-1-81. Baboon ESCs injected into NOD/SCID mice formed teratomas with all 3 germ layers. In addition, embryoid body-like spherical structures were derived and initial outgrowth was observed when embedded into extracellular matrix Matrigel. The ESC lines established in this NHP model have the potential to extend our knowledge in the fields of developmental biology, regenerative medicine, and future applications, including preclinical safety assessment of in vivo stem cell therapy.

Insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 mediate TGF-β- and myostatin-induced suppression of proliferation in porcine embryonic myogenic cell cultures

International Nuclear Information System (INIS)

Kamanga-Sollo, E.; Pampusch, M.S.; White, M.E.; Hathaway, M.R.; Dayton, W.R.

2005-01-01

We have previously shown that cultured porcine embryonic myogenic cells (PEMC) produce both insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 and secrete these proteins into their media. Exogenously added recombinant porcine (rp) IGFBP-3 and rpIGFBP-5 act via IGF-dependent and IGF-independent mechanisms to suppress proliferation of PEMC cultures. Furthermore, immunoneutralization of endogenous IGFBP-3 and IGFBP-5 in the PEMC culture medium results in increased DNA synthesis rate suggesting that endogenous IGFBP-3 and IGFBP-5 suppress PEMC proliferation. TGF-β superfamily members myostatin and TGF-β 1 have also been shown to suppress proliferation of myogenic cells, and treatment of cultured PEMC with either TGF-β 1 or myostatin significantly (P 1 and myostatin. Here, we show that immunoneutralization of IGFBP-5 also significantly (P 1 or myostatin-treated PEMC cultures restores Long-R3-IGF-I-stimulated DNA synthesis rates to 90% of the levels observed in control cultures receiving no TGF-β 1 or myostatin treatment (P 1 or myostatin-treated PEMC cultures, phosphosmad2 levels in these cultures were not affected. These findings strongly suggest that IGFBP-3 and IGFBP-5 affect processes downstream from receptor-mediated Smad phosphorylation that facilitate the ability of TGF-β and myostatin to suppress proliferation of PEMC

Effects of exposing rat embryos in utero to physical or chemical teratogens are expressed later as enhanced induction of heat-shock proteins when embryonic hearts are cultured in vitro

International Nuclear Information System (INIS)

Higo, H.; Higo, K.; Lee, J.Y.; Hori, H.; Satow, Y.

1988-01-01

In order to get more insight into the effects of teratogens on developing embryos, we investigated the protein synthesis patterns of the target organs isolated from teratogen-treated embryos. Rat embryos were either irradiated in utero with either 252Cf fission neutrons or 60Co gamma rays on day 8 of gestation or treated in utero with a bis(dichloroacetyl)diamine (a chemical teratogen) on days 9 and 10. Hearts were removed from the embryos on day 12 and were incubated in vitro at 37 degrees C in the presence of [35S]methionine for up to 8 hr. The newly synthesized labeled proteins were then analyzed qualitatively by two-dimensional polyacrylamide gel electrophoresis. Enhanced and prolonged induction of a family of heat-shock (stress) proteins with a molecular weight of about 70,000 (SP70s) was observed as compared with those of controls. Among the teratogen-treated hearts, those with gross malformations already detectable at this early stage showed especially higher inductions of SP70s than did the others. The abnormal expression of SP70s observed in the present study appears to be a reflection of persisting cellular (tissue) damage inflicted by the teratogens, and the extent of the induction may be indicative of the degree and/or type of the damage. Such persisting defects in surviving cells, manifested by abnormal induction of SP70s in the present study, might be related to malformation of embryonic hearts

Isolated rat dental pulp cell culture and transplantation with an alginate scaffold.

Science.gov (United States)

Fujiwara, Shiro; Kumabe, Shunji; Iwai, Yasutomo

2006-05-01

Many studies have been conducted on tissue stem cells in the field of regenerative medicine, and cultured dental pulp mesenchymal cells have been reported to secrete dentin matrix. In the present study we used alginate as a scaffold to transplant subcultured rat dental-pulp-derived cells subcutaneously into the back of nude mice. We found that when beta-glycerophosphate was added to the culture medium, the mRNA of the dentin sialophosphoprotein (DSPP) gene coding dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) was expressed, and an increase in alkaline phosphatase, an early marker of odontoblast differentiation, was also demonstrated. Six weeks after implantation, subcutaneous formation of radiopaque calcified bodies was observed in situ. Immunohistochemical and fine structure studies identified expression of type I collagen, type III collagen, and DSP in the mineralizing transplants, and isolated odontoblast-like cells began to form dentin-like hard tissue formation. Scattered autolyzing apoptotic cells were also observed in the transplants. The study showed that subcultured rat dental-pulp-derived cells actively differentiate into odontoblast-like cells and induce calcification in an alginate scaffold.

In vitro formation of the Merkel cell-neurite complex in embryonic mouse whiskers using organotypic co-cultures.

Science.gov (United States)

Ishida, Kentaro; Saito, Tetsuichiro; Mitsui, Toshiyuki

2018-06-01

A Merkel cell-neurite complex is a touch receptor composed of specialized epithelial cells named Merkel cells and peripheral sensory nerves in the skin. Merkel cells are found in touch-sensitive skin components including whisker follicles. The nerve fibers that innervate Merkel cells of a whisker follicle extend from the maxillary branch of the trigeminal ganglion. Whiskers as a sensory organ attribute to the complicated architecture of the Merkel cell-neurite complex, and therefore it is intriguing how the structure is formed. However, observing the dynamic process of the formation of a Merkel cell-neurite complex in whiskers during embryonic development is still difficult. In this study, we tried to develop an organotypic co-culture method of a whisker pad and a trigeminal ganglion explant to form the Merkel cell-neurite complex in vitro. We initially developed two distinct culture methods of a single whisker row and a trigeminal ganglion explant, and then combined them. By dissecting and cultivating a single row from a whisker pad, the morphogenesis of whisker follicles could be observed under a microscope. After the co-cultivation of the whisker row with a trigeminal ganglion explant, a Merkel cell-neurite complex composed of Merkel cells, which were positive for both cytokeratin 8 and SOX2, Neurofilament-H-positive trigeminal nerve fibers and Schwann cells expressing Nestin, SOX2 and SOX10 was observed via immunohistochemical analyses. These results suggest that the process for the formation of a Merkel cell-neurite complex can be observed under a microscope using our organotypic co-culture method. © 2018 Japanese Society of Developmental Biologists.

Stiff person syndrome associated anti-amphiphysin antibodies reduce GABA associated [Ca(2+)]i rise in embryonic motoneurons.

Science.gov (United States)

Geis, C; Beck, M; Jablonka, S; Weishaupt, A; Toyka, K V; Sendtner, M; Sommer, C

2009-10-01

Autoantibodies to the synaptic protein amphiphysin play a crucial pathogenic role in paraneoplastic stiff-person syndrome. Impairment of GABAergic inhibition is the presumed pathophysiological mechanism by which these autoantibodies become pathogenic. Here we used calcium imaging on rat embryonic motor neurons to investigate whether antibodies to amphiphysin directly hinder GABAergic signaling. We found that the immunoglobulin G fraction from a patient with stiff-person syndrome, containing high titer antibodies to amphiphysin and inducing stiffness in rats upon passive transfer, reduced GABA-induced calcium influx in embryonic motor neurons. Depletion of the anti-amphiphysin fraction from the patient's IgG by selective affinity chromatography abolished this effect, showing its specificity for amphiphysin. Quantification of the surface expression of the Na(+)/K(+)/2Cl(2-) cotransporter revealed a reduction after incubation with anti-amphiphysin IgG, which is concordant with a lower intracellular chloride concentration and thus impairment of GABA mediated calcium influx. Thus, anti-amphiphysin antibodies exert a direct effect on GABA signaling, which is likely to contribute to the pathogenesis of SPS.

Insulin: its binding to specific receptors and its stimulation of DNA synthesis and 2',3'-cyclic nucleotide phosphohydrolase in embryonic mouse brain cell cultures

International Nuclear Information System (INIS)

Shanker, G.; Pieringer, R.A.

1986-01-01

Previously, the authors demonstrated that ornithine decarboxylase was stimulated by insulin in cultures of embryonic mouse brain cells. In the present work, they have investigated the presence and specificity of insulin receptors in these cultures. A time study showed that maximum binding of 125 [I] labelled insulin was around 75 min. Other studies measured the influence of concentration and age on insulin binding. A displacement study using increasing concentrations of cold insulin, glucagon or growth hormone demonstrated that the specificity of the receptors for insulin was rather high. It was also found that insulin displayed a clear dose-dependent stimulation of thymidine incorporation into the brain cells. Insulin also stimulated the glial enzyme 2':3'-cyclic nucleotide phosphohydrolase (CNP-ase). The results suggest a dual role for insulin; it regulates both cell proliferation as well as differentiation

Optimal Population of Embryonic Stem Cells in "Hanging Drop" Culture for in-vitro Differentiation to Cardiac Myocytes

OpenAIRE

MIWA, Keiko; LEE, Jong-Kook; HIDAKA, Kyoko; SHI, Rong-qian; MORISAKI, Takayuki; KODAMA, Itsuo

2002-01-01

Pluripotent embryonic stem (ES) cells differentiate to cardiac myocytes in vitro by many other previous reports demonstrated "hanging-drop" method. In this study, the number of ES cells in each hanging-drop plays an important role in the cultivation of cardiac myocytes. We examined the optimal hanging-drop size to obtain embryonic stem cell-derived cardiac cells (ESCMs) in vitro using specific labeled mouse ES cells (hCGP7) which were stably transfected with the enhanced green fluorescent pro...

The effects of donor stage on the survival and function of embryonic striatal grafts in the adult rat brain; II. Correlation between positron emission tomography and reaching behaviour

International Nuclear Information System (INIS)

Dunnett, S.B.; Brooks, D.J.; Ashworth, S.; Opacka-Juffrey, J.; Myers, R.; Hume, S.P.; Torres, E.M.; Fricker, R.A.

1997-01-01

Grafts of embryonic striatal primordia are able to elicit behavioural recovery in rats which have received an excitotoxic lesion to the striatum, and it is believed that the P zones or striatal-like tissue within the transplants play a crucial role in these functional effects. We performed this study to compare the effects of different donor stage of embryonic tissue on both the morphology (see accompanying paper) and function of striatal transplants. Both the medial and lateral ganglionic eminence was dissected from rat embryos of either 10 mm, 15 mm, 19 mm, or 23 mm crown-rump length, and implanted as a cell suspension into adult rats which had received an ibotenic acid lesion 10 days prior to transplantation. After four months the animals were tested on the 'staircase task' of skilled forelimb use. At 10-14 months rats from the groups which had received grafts from 10 mm or 15 mm donor embryos were taken for positron emission tomography scanning in a small diameter postiron emission tomography scanner, using ligands to the dopamine D 1 and D 2 receptors, [ 11 C]SCH 23390 and [ 11 C]raclopride, respectively. A lesion-alone group was also scanned with the same ligands for comparison. Animals which had received transplants from the 10 mm donors showed a significant recovery with their contralateral paw on the 'staircase test'. No other groups showed recovery on this task. Similarly, the animals with grafts from the youngest donors showed a significant increase in D 1 and D 2 receptor binding when compared to the lesion-alone group. No increase in signal was observed with either ligand in the group which had received grafts from 15 mm donors. Success in paw reaching showed a strong correlation to both the positron emission tomography signal obtained and the P zone volume of the grafts.These results suggest that striatal grafts from younger donors (10 mm CRL) give greater behavioural recovery than grafts prepared from older embryos. This recovery is due to both the

Thromboxane A(2 receptor stimulation promotes closure of the rat ductus arteriosus through enhancing neointima formation.

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Tomohiro Yokota

Full Text Available Ductus arteriosus (DA closure follows constriction and remodeling of the entire vessel wall. Patent ductus arteriosus occurs when the DA does not close after birth, and this condition is currently treated using cyclooxygenase inhibitors. However, the efficacy of cyclooxygenase inhibitors is often limited. Our previous study demonstrated that low-dose thromboxane A2 receptor (TP stimulation constricted the DA with minimal adverse effects in rat neonates. However, its effect on DA remodeling remains unknown. In this study, we focused on the impact of the exogenous TP stimulation on the DA remodeling, especially intimal thickening. Using DA explants from rat fetuses at embryonic day 19 as a ex vivo model and primary cultured rat DA smooth muscle cells from embryonic day 21 as a in vitro model, we evaluated the effect of TP stimulation on the DA remodeling. The selective TP agonists U46619 and I-BOP promoted neointima formation in the ex vivo DA explants, and TP stimulation increased DA SMC migration in a dose-dependent manner. Both effects were inhibited by the selective TP antagonist SQ29548 or the siRNA against TP. TP stimulation also increased DA SMC proliferation in the presence of 10% fetal bovine serum. LC/MS/MS analysis revealed that TP stimulation increased secretion of several extracellular matrix proteins that may contribute to an increase in neointima formation. In conclusion, we uncovered that exogenous administration of TP agonist promotes neointima formation through the induction of migration and proliferation of DA SMC, which could contribute to DA closure and also to its vasoconstrictive action.

All-trans retinoic acid promotes neural lineage entry by pluripotent embryonic stem cells via multiple pathways

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Fang Bo

2009-07-01

Full Text Available Abstract Background All-trans retinoic acid (RA is one of the most important morphogens with pleiotropic actions. Its embryonic distribution correlates with neural differentiation in the developing central nervous system. To explore the precise effects of RA on neural differentiation of mouse embryonic stem cells (ESCs, we detected expression of RA nuclear receptors and RA-metabolizing enzymes in mouse ESCs and investigated the roles of RA in adherent monolayer culture. Results Upon addition of RA, cell differentiation was directed rapidly and exclusively into the neural lineage. Conversely, pharmacological interference with RA signaling suppressed this neural differentiation. Inhibition of fibroblast growth factor (FGF signaling did not suppress significantly neural differentiation in RA-treated cultures. Pharmacological interference with extracellular signal-regulated kinase (ERK pathway or activation of Wnt pathway effectively blocked the RA-promoted neural specification. ERK phosphorylation was enhanced in RA-treated cultures at the early stage of differentiation. Conclusion RA can promote neural lineage entry by ESCs in adherent monolayer culture systems. This effect depends on RA signaling and its crosstalk with the ERK and Wnt pathways.

From engineering to editing the rat genome.

Science.gov (United States)

Meek, Stephen; Mashimo, Tomoji; Burdon, Tom

2017-08-01

Since its domestication over 100 years ago, the laboratory rat has been the preferred experimental animal in many areas of biomedical research (Lindsey and Baker The laboratory rat. Academic, New York, pp 1-52, 2006). Its physiology, size, genetics, reproductive cycle, cognitive and behavioural characteristics have made it a particularly useful animal model for studying many human disorders and diseases. Indeed, through selective breeding programmes numerous strains have been derived that are now the mainstay of research on hypertension, obesity and neurobiology (Okamoto and Aoki Jpn Circ J 27:282-293, 1963; Zucker and Zucker J Hered 52(6):275-278, 1961). Despite this wealth of genetic and phenotypic diversity, the ability to manipulate and interrogate the genetic basis of existing phenotypes in rat strains and the methodology to generate new rat models has lagged significantly behind the advances made with its close cousin, the laboratory mouse. However, recent technical developments in stem cell biology and genetic engineering have again brought the rat to the forefront of biomedical studies and enabled researchers to exploit the increasingly accessible wealth of genome sequence information. In this review, we will describe how a breakthrough in understanding the molecular basis of self-renewal of the pluripotent founder cells of the mammalian embryo, embryonic stem (ES) cells, enabled the derivation of rat ES cells and their application in transgenesis. We will also describe the remarkable progress that has been made in the development of gene editing enzymes that enable the generation of transgenic rats directly through targeted genetic modifications in the genomes of zygotes. The simplicity, efficiency and cost-effectiveness of the CRISPR/Cas gene editing system, in particular, mean that the ability to engineer the rat genome is no longer a limiting factor. The selection of suitable targets and gene modifications will now become a priority: a challenge where

Gel entrapment culture of rat hepatocytes for investigation of tetracycline-induced toxicity

International Nuclear Information System (INIS)

Shen Chong; Meng Qin; Schmelzer, Eva; Bader, Augustinus

2009-01-01

This paper aimed to explore three-dimensionally cultured hepatocytes for testing drug-induced nonalcoholic steatohepatitis. Gel entrapped rat hepatocytes were applied for investigation of the tetracycline-induced steatohepatitis, while hepatocyte monolayer was set as a control. The toxic responses of hepatocytes were systematically evaluated by measuring cell viability, liver-specific function, lipid accumulation, oxidative stress, adenosine triphosphate content and mitochondrial membrane potential. The results suggested that gel entrapped hepatocytes showed cell death after 96 h of tetracycline treatment at 25 μM which is equivalent to toxic serum concentration in rats, while hepatocyte monolayer showed cell death at a high dose of 200 μM. The concentration-dependent accumulation of lipid as well as mitochondrial damage were regarded as two early events for tetracycline hepatotoxicity in gel entrapment culture due to their detectability ahead of subsequent increase of oxidative stress and a final cell death. Furthermore, the potent protection of fenofibrate and fructose-1,6-diphosphate were evidenced in only gel entrapment culture with higher expressions on the genes related to β-oxidation than hepatocyte monolayer, suggesting the mediation of lipid metabolism and mitochondrial damage in tetracycline toxicity. Overall, gel entrapped hepatocytes in three-dimension reflected more of the tetracycline toxicity in vivo than hepatocyte monolayer and thus was suggested as a more relevant system for evaluating steatogenic drugs.

Optimization of culture conditions to support long-term self-renewal of buffalo (Bubalus bubalis) embryonic stem cell-like cells.

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Sharma, Ruchi; George, Aman; Kamble, Nitin Manchindra; Singh, Karn Pratap; Chauhan, Manmohan Singh; Singla, Suresh Kumar; Manik, Radhey Sham; Palta, Prabhat

2011-12-01

A culture system capable of sustaining self-renewal of buffalo embryonic stem (ES) cell-like cells in an undifferentiated state over a long period of time was developed. Inner cell masses were seeded on KO-DMEM+15% KO-serum replacer on buffalo fetal fibroblast feeder layer. Supplementation of culture medium with 5 ng/mL FGF-2 and 1000 IU/mL mLIF gave the highest (p<0.05) rate of primary colony formation. The ES cell-like cells' colony survival rate and increase in colony size were highest (p<0.05) following supplementation with FGF-2 and LIF compared to other groups examined. FGF-2 supplementation affected the quantitative expression of NANOG, SOX-2, ACTIVIN A, BMP 4, and TGFβ1, but not OCT4 and GREMLIN. Supplementation with SU5402, an FGFR inhibitor (≥20 μM) increased (p<0.05) the percentage of colonies that differentiated. FGFR1-3 and ERK1, K-RAS, E-RAS, and SHP-2, key signaling intermediates of FGF signaling, were detected in ES cell-like cells. Under culture conditions described, three ES cell lines were derived that, to date, have been maintained for 135, 95, and 85 passages for over 27, 19, and 17 months, respectively, whereas under other conditions examined, ES cell-like cells did not survive beyond passage 10. The ES cell-like cells were regularly monitored for expression of pluripotency markers and their potency to form embryoid bodies.

Regulation of pro-adrenocorticotropin-endorphin synthesis and secretion in cultured neonatal rat anterior pituitary

Energy Technology Data Exchange (ETDEWEB)

Sato, S.M.; Mains, R.E. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA))

1987-08-01

Previous work demonstrated that newborn rat anterior pituitary corticotropes display processing patterns for pro-ACTH/endorphin that are different from the adult. The synthesis and release of beta-endorphin-related peptides was examined in dispersed cell and explant cultures of newborn anterior pituitary to investigate corticotrope development further. The temporal pattern of pro-ACTH/endorphin processing differed significantly from adult rat melanotropes and AtT-20 cells. While pro-ACTH/endorphin processing begins within 30 min of synthesis in adult melanotropes and AtT-20 cells, pulse-labeling of newborn corticotropes in culture indicated that pro-ACTH/endorphin remained uncleaved for at least 90 min after synthesis. With further incubation, there was a decrease in radioactivity associated with the precursor and an equivalent rise in the radioactivity associated with beta-endorphin and beta-lipotropin. However, unprocessed precursor still remained in the cultured newborn anterior pituitary cells after a 25-h chase. Although intact pro-ACTH/endorphin from newborn corticotropes was very long-lived, the precursor did undergo oligosaccharide maturation and became endoglycosidase H resistant within 1 h after synthesis. Similar to the adult, pro-ACTH/endorphin synthesis was doubled in cultures of newborn anterior pituitary chronically treated with 10 nM CRF resulting in a 3- to 4-fold stimulation of secretion over the basal rate. However, unlike the AtT-20 cell or adult rat corticotrope, the proteolytic processing of pro-ACTH/endorphin in the newborn corticotrope was altered by chronic secretagogue treatment; less pro-ACTH/endorphin was converted to beta-endorphin in secretagogue-treated corticotropes than in controls. Thus processing of pro-ACTH/endorphin in the corticotrope is not mature by birth and can be regulated by chronic CRF treatment.

Regulation of pro-adrenocorticotropin-endorphin synthesis and secretion in cultured neonatal rat anterior pituitary

International Nuclear Information System (INIS)

Sato, S.M.; Mains, R.E.

1987-01-01

Previous work demonstrated that newborn rat anterior pituitary corticotropes display processing patterns for pro-ACTH/endorphin that are different from the adult. The synthesis and release of beta-endorphin-related peptides was examined in dispersed cell and explant cultures of newborn anterior pituitary to investigate corticotrope development further. The temporal pattern of pro-ACTH/endorphin processing differed significantly from adult rat melanotropes and AtT-20 cells. While pro-ACTH/endorphin processing begins within 30 min of synthesis in adult melanotropes and AtT-20 cells, pulse-labeling of newborn corticotropes in culture indicated that pro-ACTH/endorphin remained uncleaved for at least 90 min after synthesis. With further incubation, there was a decrease in radioactivity associated with the precursor and an equivalent rise in the radioactivity associated with beta-endorphin and beta-lipotropin. However, unprocessed precursor still remained in the cultured newborn anterior pituitary cells after a 25-h chase. Although intact pro-ACTH/endorphin from newborn corticotropes was very long-lived, the precursor did undergo oligosaccharide maturation and became endoglycosidase H resistant within 1 h after synthesis. Similar to the adult, pro-ACTH/endorphin synthesis was doubled in cultures of newborn anterior pituitary chronically treated with 10 nM CRF resulting in a 3- to 4-fold stimulation of secretion over the basal rate. However, unlike the AtT-20 cell or adult rat corticotrope, the proteolytic processing of pro-ACTH/endorphin in the newborn corticotrope was altered by chronic secretagogue treatment; less pro-ACTH/endorphin was converted to beta-endorphin in secretagogue-treated corticotropes than in controls. Thus processing of pro-ACTH/endorphin in the corticotrope is not mature by birth and can be regulated by chronic CRF treatment

Postnatal fate of the ultimobranchial remnants in the rat thyroid gland.

Science.gov (United States)

Vázquez-Román, Victoria; Utrilla, José C; Fernández-Santos, José M; Conde, Esperanza; Bernabé, Reyes; Sampedro, Consuelo; Martín-Lacave, Inés

2013-07-01

The ultimobranchial follicles (UBFs) are considered embryonic remnants from the ultimobranchial body (UBB). They are follicular structures that vary in size and appearance depending on the age of the rat. The main objective of this article was to study the progressive changes in shape, size, and frequency of the UBFs in the postnatal rat, from birth to old-age. To accomplish that objective, a systematic morphometric and incidental study of the UBF has been carried out in 110 Wistar rats of different ages and both sexes, divided into three groups: 1) young rats (5-90-day-old); 2) adult rats (6-15-month-old), and 3) old rats (18-24-month-old). The glands were serially sectioned and immunostained for calcitonin at five equidistant levels. According to our results, UBFs were observed in all thyroid glands but a more exhaustive sampling was occasionally necessary in male rats. In young rats, immature UBFs predominantly appeared whereas in adult rats, mature UBFs with cystic appearance and variable luminal content prevailed. We frequently found spontaneous anomalous UBFs in old rats, which we have termed as "ultimobranchial cystadenomata." Additionally, in young rats, UBF areas significantly increased with age and they were larger when compared to that of normal thyroid follicles. Likewise, in adult rats, UBFs were significantly larger than normal thyroid follicles but only in female rats. In general, UBFs in females were also significantly larger than those found in male rats. Finally, all these differences related to UBFs together with a higher incidence in females of UB cystadenomata suggest a sexual dimorphism in regard to the destiny of these embryonic remnants during postnatal thyroid development. Copyright © 2013 Wiley Periodicals, Inc.

Extracellular matrix components influence DNA synthesis of rat hepatocytes in primary culture

International Nuclear Information System (INIS)

Sawada, N.; Tomomura, A.; Sattler, C.A.; Sattler, G.L.; Kleinman, H.K.; Pitot, H.C.

1986-01-01

The effects of several extracellular matrix components (EMCs) - fibronectin (Fn), laminin (Ln), type I (C-I) and type IV (C-IV) collagen - on DNA synthesis in rat hepatocytes in primary culture were examined by both quantitative scintillation spectrometry and autoradiography of [ 3 H]thymidine incorporation. Hepatocytes cultured on Fn showed the most active DNA synthesis initiated by epidermal growth factor (EGF) with decreasing levels of [ 3 H]thymidine uptake exhibited in the cell cultured on C-IV, C-I, and Ln, respectively. The decreasing level of DNA synthesis in hepatocytes cultured on Fn, C-IV, C-I, and Ln respectively was not influenced by cell density. The number of EGF receptors of hepatocytes was also not influenced by EMCs. These data suggest that EMCs modify hepatocyte DNA synthesis by means of post-EGF-receptor mechanisms which are regulated by both growth factors and cell density

Effect of retinoic acid on midkine gene expression in rat anterior pituitary cells.

Science.gov (United States)

Maliza, Rita; Fujiwara, Ken; Azuma, Morio; Kikuchi, Motoshi; Yashiro, Takashi

2017-06-29

Retinoic acid (RA) is converted from retinal by retinaldehyde dehydrogenases (RALDHs) and is an essential signaling molecule in embryonic and adult tissue. We previously reported that RALDH1 was produced in the rat anterior pituitary gland and hypothesized that RA was generated in the gland. Midkine (MK) is an RA-inducible growth factor, and MK production in the rat anterior pituitary gland was recently reported. However, the mechanism that regulates gene expression of MK in the pituitary gland has not been determined. To investigate regulation of MK production in the anterior pituitary gland, we analyzed changes in MK mRNA in cultured rat anterior pituitary cells. We identified MK-expressing cells by double-staining with in situ hybridization and immunohistochemical techniques for RALDH1. MK mRNA was expressed in RALDH1-producing cells in the anterior pituitary gland. Using isolated anterior pituitary cells of rats, we examined the effect of RA on gene expression of MK. Quantitative real-time PCR revealed that 72 h exposure to a concentration of 10 -6 M of retinal and all-trans retinoic acid increased MK mRNA levels by about 2-fold. Moreover, the stimulatory effect of all-trans retinoic acid was mimicked by the RA receptor agonist Am80. This is the first report to show that RA is important in regulating MK expression in rat anterior pituitary gland.

Distinct angiotensin II receptor in primary cultures of glial cells from rat brain

International Nuclear Information System (INIS)

Raizada, M.K.; Phillips, M.I.; Crews, F.T.; Sumners, C.

1987-01-01

Angiotensin II (Ang-II) has profound effects on the brain. Receptors for Ang-II have been demonstrated on neurons, but no relationship between glial cells and Agn-II has been established. Glial cells (from the hypothalamus and brain stem of 1-day-old rat brains) in primary culture have been used to demonstrate the presence of specific Ang-II receptors. Binding of 125 I-Ang-II to glial cultures was rapid, reversible, saturable, and specific for Ang-II. The rank order of potency of 125 I-Ang-II binding was determined. Scatchard analysis revealed a homogeneous population of high-affinity binding sites with a B/sub max/ of 110 fmol/mg of protein. Light-microscopic autoradiography of 125 I-Ang-II binding supported the kinetic data, documenting specific Ang-II receptors on the glial cells. Ang-II stimulated a dose-dependent hydrolysis of phosphatidylinositols in glial cells, an effect mediated by Ang-II receptors. However, Ang-II failed to influence [ 3 H] norepinephrine uptake, and catecholamines failed to regulate Ang-II receptors, effects that occur in neurons. These observations demonstrate the presence of specific Ang-II receptors on the glial cells in primary cultures derived from normotensive rat brain. The receptors are kinetically similar to, but functionally distinct from, the neuronal Ang-II receptors

Comparison of radiometric and conventional culture systems in detecting Haemophilus influenzae type b bacteremia in rats

International Nuclear Information System (INIS)

Mitchell, M.J.; Zwahlen, A.; Elliott, H.L.; Ford, N.K.; Charache, F.P.; Moxon, E.R.

1985-01-01

To compare the efficiency of detecting Haemophilus influenzae type b bacteremia by the BACTEC radiometric system and a conventional Trypticase soy broth blood culture system, the authors developed an in vivo model of bacteremia in rats. After intravenous injection of 50 to 200 CFU into adult rats, there was a linear logarithmic increase in CFU per milliliter of rat blood during the first 10 h (r = 0.98), allowing accurate prediction of the level of bacteremia with time. Culture bottles were inoculated with 0.5 ml of blood obtained by cardiac puncture and processed as clinical samples in the microbiology laboratory with RS and conventional protocols. They found the following. (i) The first detection of bacteremia by RS was similar to that by TSB if a Gram stain of the TSB was done on day 1 and was superior if that smear was omitted (P less than 0.01). (ii) The detection times in both systems were comparable at different magnitudes of bacteremia (10(1) to 10(4) CFU/ml). (iii) Supplementation of inoculated bottles with 2 ml of sterile rat blood interfered with Gram stain detection in TSB but resulted in increased 14 CO 2 production in RS. (iv) No difference in detection time was found between RS and TSB for four different clinical isolates. These studies show that, in a biologically relevant model, the detection of positive blood cultures for H. influenzae type b by RS was comparable to or better than detection by TSB when blood was processed analogously to clinical specimens

Insulin growth factors regulate the mitotic cycle in cultured rat sympathetic neuroblasts

International Nuclear Information System (INIS)

DiCicco-Bloom, E.; Black, I.B.

1988-01-01

While neuronal mitosis is uniquely restricted to early development, the underlying regulation remains to be defined. The authors have now developed a dissociated, embryonic sympathetic neuron culture system that uses fully defined medium in which cells enter the mitotic cycle. The cultured cells expressed two neuronal traits, tyrosine hydroxylase and the neuron-specific 160-kDa neurofilament subunit protein, but were devoid of glial fibrillary acidic protein, a marker for non-myelin-forming Schwann cells in ganglia. Approximately one-third of the tyrosine hydroxylase-positive cells synthesized DNA in culture, specifically incorporating [ 3 H]thymidine into their nuclei. They used this system to define factors regulating the mitotic cycle in sympathetic neuroblasts. Members of the insulin family of growth factors, including insulin and insulin-like growth factors I and II, regulated DNA synthesis in the presumptive neuroblasts. Insulin more than doubled the proportion of tyrosine hydroxylase-positive cells entering the mitotic cycle, as indicated by autoradiography of [ 3 H]thymidine incorporation into nuclei. Scintillation spectrometry was an even more sensitive index of DNA synthesis. In contrast, the trophic protein nerve growth factor exhibited no mitogenic effect, suggesting that the mitogenic action of insulin growth factors is highly specific. The observations are discussed in the context of the detection of insulin growth factors and receptors in the developing brain

Carbon Tetrachloride Increases Intracellular Calcium in Rat Liver and Hepatocyte Cultures

Science.gov (United States)

1986-05-12

tible to destruction by CC14 ( Head ~ al., 1981). Thus, the activation of CC14 to a toxic moiety clearly depends upon metabolism by one or more... embryologic development (Wyllie, 1986). In contrast, Toyo-oka et al. (1985) could not establish that phospholipase& or proteases were involved in ischemic...D. M. Bissell, and u. A Meyer. (1977) Drug Metabolism in Adult Rat Hepatocyte& in Primary Monolayer Culture. Gastroenterology 72:1232-1239. Head , B

The hTH-GFP reporter rat model for the study of Parkinson's disease.

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Lorraine Iacovitti

Full Text Available Parkinson disease (PD is the second leading neurodegenerative disease in the US. As there is no known cause or cure for PD, researchers continue to investigate disease mechanisms and potential new therapies in cell culture and in animal models of PD. In PD, one of the most profoundly affected neuronal populations is the tyrosine hydroxylase (TH-expressing dopaminergic (DA neurons of the substantia nigra pars compacta (SNpc. These DA-producing neurons undergo degeneration while neighboring DA-producing cells of the ventral tegmental area (VTA are largely spared. To aid in these studies, The Michael J. Fox Foundation (MJFF partnered with Thomas Jefferson University and Taconic Inc. to generate new transgenic rat lines carrying the human TH gene promoter driving EGFP using a 11 kb construct used previously to create a hTH-GFP mouse reporter line. Of the five rat founder lines that were generated, three exhibited high level specific GFP fluorescence in DA brain structures (ie. SN, VTA, striatum, olfactory bulb, hypothalamus. As with the hTH-GFP mouse, none of the rat lines exhibit reporter expression in adrenergic structures like the adrenal gland. Line 12141, with its high levels of GFP in adult DA brain structures and minimal ectopic GFP expression in non-DA structures, was characterized in detail. We show here that this line allows for anatomical visualization and microdissection of the rat midbrain into SNpc and/or VTA, enabling detailed analysis of midbrain DA neurons and axonal projections after toxin treatment in vivo. Moreover, we further show that embryonic SNpc and/or VTA neurons, enriched by microdissection or FACS, can be used in culture or transplant studies of PD. Thus, the hTH-GFP reporter rat should be a valuable tool for Parkinson's disease research.

Embryonic stem cells in pig and cattle

DEFF Research Database (Denmark)

Maddox-Hyttel, Poul; Wolf, Xenia Asbæk; Rasmussen, Mikkel Aabech

2007-01-01

Porcine and bovine cell lines derived from the inner cell mass (ICM) or epiblasts of blastocysts have been maintained over extended periods of time and characterized by morphology, identification of some stem cell markers and, in few cases, by production of chimaeric offspring. However, germ line...... transmission in chimaeras has never been obtained. Due to this incomplete characterization of the cell lines, the expression embryonic stem (ES)-like cells is presently used in pig and cattle. The ICM or epiblast can be isolated from the blastocyst by whole blastocyst culture, mechanical isolation......, or immunosurgery, and they are generally cultured on feeder cells. The resulting ES-like cells may be differentiated in vivo by chimaera and teratoma formation or in vitro by embryoid body formation and monolayer induction. It is likely that more well characterized and stable porcine and bovine ES cell lines...

Tumor-associated proteins in rat submandibular gland induced by DMBA and irradiation

International Nuclear Information System (INIS)

Oh, Sung Ook; Choi, Soon Chul; Park, Tae Won; You, Dong Soo

1997-01-01

This study was performed in order to identify changes of the plasma membrane proteins in rat submandibular gland tumors induced by 7,12-dimethylbenz[a]anthracene [DMBA] and X-irradiation. Two kinds of tumor associated membrane proteins (protein A and B) were isolated with 3 M KCl extraction from rat submandibular gland tumors induced by DMBA and X-irradiation. To identify their antigenicities, immunoelectrophoresis and double immunodiffusion was carried out with various proteins extracted from liver, heart, skin and pancreas of adult rats and from embryonic liver, heart and skin. The rabbit antisera against the protein A did not cross-react with any of the proteins extracted from the above mentioned tissues, suggesting that protein A might be tumor specific antigen. However, the rabbit antisera against protein B was precipitated with proteins extracted from the liver of adult and embryonic rats. Polyacrylamide gel electrophoresis of these two proteins (A and B) showed that protein A was a dimer with molecular weights of 69,000 and 35,000 dalton, whereas protein B was a monomer with molecular weight of 50,000 dalton.

β-adrenergic receptor binding characteristics and responsiveness in cultured Wistar-Kyoto rat arterial smooth muscle cells

International Nuclear Information System (INIS)

Jazayeri, A.; Meyer, W.J. III

1988-01-01

The tone of arterial blood vessels is regulated by the catecholamines through their receptors on arterial smooth muscle cells (ASMC). β- 2 -adrenergic receptors of ASMC mediate vasodilation through agonist mediated c-AMP production. Previous reports have described these receptors on freshly isolated blood vessels. This study demonstrates the presence of β 2 -adrenergic receptors on cultured rat ASMC and that these receptors are functional. β-adrenergic receptor binding was measured using [ 3 H]-dihydroalprenolol (DHA) binding to the membrane of cultured ASMC from normotensive Wistar-Kyoto rats. The ASMC β-adrenergic receptors have a Kd of 0.56 +/- 0.16 nM and a Bmax of 57.2 +/- 21.7 fmol/mg protein. Competition binding studies revealed a much greater affinity of these receptors for epinephrine than norepinephrine, indicating the preponderance of a β 2 -adrenergic receptor subtype. Isoproterenol stimulation of cultured ASMC resulted in a 14 +/- 7 fold increase in intracellular c-AMP content of these cells indicating these receptors are functional. β-adrenergic receptors of cultured ASMC provide an excellent system in which the association between hypertension and observed β-adrenergic receptor differences can be further explored

Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

International Nuclear Information System (INIS)

Scott, C.D.; Baxter, R.C.

1987-01-01

Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [ 125 I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density

Functional cooperativity between two TPA responsive elements in undifferentiated F9 embryonic stem cells.

OpenAIRE

Okuda, A; Imagawa, M; Sakai, M; Muramatsu, M

1990-01-01

We have recently identified an enhancer, termed GPEI, in the 5'-flanking region of the rat glutathione transferase P gene, that is composed of two imperfect TPA (phorbol 12-O-tetradecanoate 13-acetate) responsive elements (TREs). Unlike other TRE-containing enhancers, GPEI exhibits a strong transcriptional enhancing activity in F9 embryonic stem cells. Mutational analyses have revealed that the high activity of GPEI is mediated by two imperfect TREs. Each TRE-like sequence has no activity by ...

Embryonic stem-like cells from rabbit blastocysts cultured with melatonin could differentiate into three germ layers in vitro and in vivo.

Science.gov (United States)

Wei, Ruxue; Zhao, Xueming; Hao, Haisheng; Du, Weihua; Zhu, Huabin

2016-11-01

The rabbit is considered an important model animal from which to obtain embryonic stem cells because of the utility of this animal in physiology and reproductive research. Here, we derived rabbit ES-like (rES-like) cells from blastocysts of superovulated Japanese white rabbits using culture medium containing 10 -7  M melatonin, 10 ng/mL basic fibroblast growth factor, and 1,000 IU/mL human leukemia inhibitory factor. This concentration of melatonin had the most significant positive effects on the proliferation inner cell mass-derived cells (improving rates from 19.97% to 34.57%) and the longevity of passaging rES-like cells. Melatonin also enhanced the expression of pluripotent genes-including alkaline phosphatase, Pou5f1, Sox2, Klf4, c-Myc, Nanog, Line28a, and surface marker proteins-in fifth-passage rES-like cells. In vitro, these rES-like cells could spontaneously differentiate into some somatic cells, such as beating cardiomyocytes; formed embryoid bodies; expressed markers of the three germ layers after differentiation; and formed teratomas after injection into non-obese diabetic-severe combined immune deficient (NOD-SCID) mice. Thus, melatonin helped coax ES-like cells from rabbit blastocysts, which raises intriguing questions about the relationship between pluripotency and proliferation in rabbit embryonic stem cells. Mol. Reprod. Dev. 83: 1003-1014, 2016 © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Early embryonic development and transplantation in tree shrews

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Lan-Zhen YAN

2016-07-01

Full Text Available As a novel experimental animal model, tree shrews have received increasing attention in recent years. Despite this, little is known in regards to the time phases of their embryonic development. In this study, surveillance systems were used to record the behavior and timing of copulations; embryos at different post-copulation stages were collected and cultured in vitro; and the developmental characteristics of both early-stage and in vitro cultured embryos were determined. A total of 163 females were collected following effective copulation, and 150 were used in either unilateral or bilateral oviduct embryo collections, with 307 embryos from 111 females obtained (conception rate=74%. Among them, 237 embryos were collected from 78 females, bilaterally, i.e., the average embryo number per female was 3.04; 172 fertilized eggs collected from 55 females, bilaterally, were cultured for 24-108 h in vitro for developmental observations; finally, 65 embryos from 23 bilateral cases and 70 embryos from 33 unilateral cases were used in embryo transplantation.

Cytosolic labile zinc: a marker for apoptosis in the developing rat brain.

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Lee, Joo-Yong; Hwang, Jung Jin; Park, Mi-Ha; Koh, Jae-Young

2006-01-01

Cytosolic zinc accumulation was thought to occur specifically in neuronal death (necrosis) following acute injury. However, a recent study demonstrated that zinc accumulation also occurs in adult rat neurons undergoing apoptosis following target ablation, and in vitro experiments have shown that zinc accumulation may play a causal role in various forms of apoptosis. Here, we examined whether intraneuronal zinc accumulation occurs in central neurons undergoing apoptosis during development. Embryonic and newborn Sprague-Dawley rat brains were double-stained for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) detection of apoptosis and immunohistochemical detection of stage-specific neuronal markers, such as nestin, proliferating cell nuclear antigen (PCNA), TuJ1 and neuronal nuclear specific protein (NeuN). The results revealed that apoptotic cell death occurred in neurons of diverse stages (neural stem cells, and dividing, young and adult neurons) throughout the brain during the embryonic and early postnatal periods. Further staining of brain sections with acid fuchsin or zinc-specific fluorescent dyes showed that all of the apoptotic neurons were acidophilic and contained labile zinc in their cell bodies. Cytosolic zinc accumulation was also observed in cultured cortical neurons undergoing staurosporine- or sodium nitroprusside (SNP)-induced apoptosis. In contrast, zinc chelation with CaEDTA or N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) reduced SNP-induced apoptosis but not staurosporine-induced apoptosis, indicating that cytosolic zinc accumulation does not play a causal role in all forms of apoptosis. Finally, the specific cytosolic zinc accumulation may have a practical application as a relatively simple marker for neurons undergoing developmental apoptosis.

Msx-2 expression and glucocorticoid-induced overexpression in embryonic mouse submandibular glands.

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Jaskoll, T; Luo, W; Snead, M L

1998-01-01

It is well known that the process of branching morphogenesis requires epithelial-mesenchymal interactions. One outstanding model for the study of tissue interactions during branching morphogenesis is the embryonic mouse submandibular gland (SMG). Although it has been clearly demonstrated that the branching pattern is dependent on interactions between the epithelium and the surrounding mesenchyme, little is known about the molecular mechanism underlying the branching process. One group of transcription factors that likely participates in the control of epithelial-mesenchymal inductive interactions are the Msx-class of homeodomain-containing proteins. In this paper, we focus on Msx-2 because its developmental expression is correlated with inductive interactions, suggesting that Msx-2 may play a functional role during cell-cell interactions. We demonstrate the expression of Msx-2 mRNA and protein to be primarily in the branching epithelia with progressive embryonic (E13 to E15) SMG development and, to a lesser extent, in the mesenchyme. We also show that Msx-2 is expressed by embryonic SMG primordia cultured under defined conditions. In addition, to begin to delineate a functional role for Msx-2, we employed an experimental strategy by using exogenous glucocorticoid (CORT) treatment of embryonic SMGs in vitro and in vivo to significantly enhance branching morphogenesis and evaluate the effect of CORT treatment on embryonic SMG Msx-2 expression. A marked increase in Msx-2 transcripts and protein is detected with in vitro and in vivo CORT treatment. Our studies indicate that one mechanism of CORT regulation of salivary gland morphogenesis is likely through the modulation of Msx-2 gene expression.

Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

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Yamano, Noriko [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Kimura, Tohru, E-mail: tkimura@patho.med.osaka-u.ac.jp [Department of Pathology, Medical School, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Watanabe-Kushima, Shoko [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Shinohara, Takashi [Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501 (Japan); Nakano, Toru, E-mail: tnakano@patho.med.osaka-u.ac.jp [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Department of Pathology, Medical School, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan)

2010-02-12

Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.

Feasibility of direct oxygenation of primary-cultured rat hepatocytes using polyethylene glycol-decorated liposome-encapsulated hemoglobin (LEH).

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Naruto, Hirosuke; Huang, Hongyun; Nishikawa, Masaki; Kojima, Nobuhiko; Mizuno, Atsushi; Ohta, Katsuji; Sakai, Yasuyuki

2007-10-01

We tested the short-term efficacy of liposome-encapsulated hemoglobin (LEH) in cultured rat hepatocytes. Supplementation with LEH (20% of the hemoglobin concentration of blood) did not lower albumin production in static culture, and completely reversed the cell death and deterioration in albumin production caused by an oxygen shortage in 2D flat-plate perfusion bioreactors.

An improved in vitro blood-brain barrier model: rat brain endothelial cells co-cultured with astrocytes.

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Abbott, N Joan; Dolman, Diana E M; Drndarski, Svetlana; Fredriksson, Sarah M

2012-01-01

In vitro blood-brain barrier (BBB) models using primary cultured brain endothelial cells are important for establishing cellular and molecular mechanisms of BBB function. Co-culturing with BBB-associated cells especially astrocytes to mimic more closely the in vivo condition leads to upregulation of the BBB phenotype in the brain endothelial cells. Rat brain endothelial cells (RBECs) are a valuable tool allowing ready comparison with in vivo studies in rodents; however, it has been difficult to obtain pure brain endothelial cells, and few models achieve a transendothelial electrical resistance (TEER, measure of tight junction efficacy) of >200 Ω cm(2), i.e. the models are still relatively leaky. Here, we describe methods for preparing high purity RBECs and neonatal rat astrocytes, and a co-culture method that generates a robust, stable BBB model that can achieve TEER >600 Ω cm(2). The method is based on >20 years experience with RBEC culture, together with recent improvements to kill contaminating cells and encourage BBB differentiation.Astrocytes are isolated by mechanical dissection and cell straining and are frozen for later co-culture. RBECs are isolated from 3-month-old rat cortices. The brains are cleaned of meninges and white matter and enzymatically and mechanically dissociated. Thereafter, the tissue homogenate is centrifuged in bovine serum albumin to separate vessel fragments from other cells that stick to the myelin plug. The vessel fragments undergo a second enzyme digestion to separate pericytes from vessels and break down vessels into shorter segments, after which a Percoll gradient is used to separate capillaries from venules, arterioles, and single cells. To kill remaining contaminating cells such as pericytes, the capillary fragments are plated in puromycin-containing medium and RBECs grown to 50-60% confluence. They are then passaged onto filters for co-culture with astrocytes grown in the bottom of the wells. The whole procedure takes ∼2

Exogenous transforming growth factor-β1 enhances smooth muscle differentiation in embryonic mouse jejunal explants.

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Coletta, Riccardo; Roberts, Neil A; Randles, Michael J; Morabito, Antonino; Woolf, Adrian S

2017-01-13

An ex vivo experimental strategy that replicates in vivo intestinal development would in theory provide an accessible setting with which to study normal and dysmorphic gut biology. The current authors recently described a system in which mouse embryonic jejunal segments were explanted onto semipermeable platforms and fed with chemically defined serum-free media. Over 3 days in organ culture, explants formed villi and they began to undergo spontaneous peristalsis. As defined in the current study, the wall of the explanted gut failed to form a robust longitudinal smooth muscle (SM) layer as it would do in vivo over the same time period. Given the role of transforming growth factor β1 (TGFβ1) in SM differentiation in other organs, it was hypothesized that exogenous TGFβ1 would enhance SM differentiation in these explants. In vivo, TGFβ receptors I and II were both detected in embryonic longitudinal jejunal SM cells and, in organ culture, exogenous TGFβ1 induced robust differentiation of longitudinal SM. Microarray profiling showed that TGFβ1 increased SM specific transcripts in a dose dependent manner. TGFβ1 proteins were detected in amniotic fluid at a time when the intestine was physiologically herniated. By analogy with the requirement for exogenous TGFβ1 for SM differentiation in organ culture, the TGFβ1 protein that was demonstrated to be present in the amniotic fluid may enhance intestinal development when it is physiologically herniated in early gestation. Future studies of embryonic intestinal cultures should include TGFβ1 in the defined media to produce a more faithful model of in vivo muscle differentiation. Copyright © 2017 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd. Copyright © 2017 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.

Ginsenoside Rb1 Protects Rat Neural Progenitor Cells against Oxidative Injury

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Na Ni

2014-03-01

Full Text Available Ginseng, the root of Panax ginseng C.A. Meyer, has been used as a tonic to enhance bodily functions against various ailments for hundreds of years in Far Eastern countries without apparent adverse effects. Ginsenoside Rb1, one of the most active ingredients of ginseng, has been shown to possess various pharmacological activities. Here we report that Rb1 exhibits potent neuroprotective effects against oxidative injury induced by tert-butylhydroperoxide (t-BHP. Lactate dehydrogenase (LDH assay demonstrated that incubation with 300 µm t-BHP for 2.5 h led to a significant cell loss of cultured rat embryonic cortex-derived neural progenitor cells (NPCs and the cell viability was pronouncedly increased by 24 h pretreatment of 10 µm Rb1. TUNEL staining further confirmed that pretreatment of Rb1 significantly reduced the cell apoptosis in t-BHP-induced oxidative injury. Real time PCR revealed that pretreatment with Rb1 activated Nrf2 pathway in cultured NPCs and led to an elevated expression of HO-1. The results of the present study demonstrate that Rb1 shows a potent anti-oxidative effect on cultured NPCs by activating Nrf2 pathway.

Mechanobiology of embryonic limb development.

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Nowlan, Niamh C; Murphy, Paula; Prendergast, Patrick J

2007-04-01

Considerable evidence exists to support the hypothesis that mechanical forces have an essential role in healthy embryonic skeletal development. Clinical observations and experimental data indicate the importance of muscle contractions for limb development. However, the influence of these forces is seldom referred to in biological descriptions of bone development, and perhaps this is due to the fact that the hypothesis that mechanical forces are essential for normal embryonic skeletal development is difficult to test and elaborate experimentally in vivo, particularly in humans. Computational modeling has the potential to address this issue by simulating embryonic growth under a range of loading conditions but the potential of such models has yet to be fully exploited. In this article, we review the literature on mechanobiology of limb development in three main sections: (a) experimental alteration of the mechanical environment, (b) mechanical properties of embryonic tissues, and (c) the use of computational models. Then we analyze the main issues, and suggest how experimental and computational fields could work closer together to enhance our understanding of mechanobiology of the embryonic skeleton.

Human amniotic epithelial cell feeder layers maintain mouse embryonic stem cell pluripotency via epigenetic regulation of the c-Myc promoter.

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Liu, Te; Cheng, Weiwei; Liu, Tianjin; Guo, Lihe; Huang, Qin; Jiang, Lizhen; Du, Xiling; Xu, Fuhui; Liu, Zhixue; Lai, Dongmei

2010-02-01

Mouse embryonic stem cells (ESCs) are typically cultured on a feeder layer of mouse embryonic fibroblasts (MEFs), with leukemia inhibitory factor (LIF) added to maintain them in an undifferentiated state. We have previously shown that human amniotic epithelial cells (hAECs) can be used as feeder cells to maintain mouse ESC pluripotency, but the mechanism for this is unknown. In the present study, we found that CpG islands 5' of the c-Myc gene remain hypomethylated in mouse ESCs cultured on hAECs. In addition, levels of acetylation of histone H3 and trimethylation of histone H3K4 in the c-Myc gene promoter were higher in ES cells cultured on hAECs than those in ES cells cultured on MEFs. These data suggested that hAECs can alter mouse ESC gene expression via epigenetic modification of c-Myc, providing a possible mechanism for the hAEC-induced maintenance of ESCs in an undifferentiated state.

Growth and morphogenesis of embryonic mouse organs on non-coated and extracellular matrix-coated Biopore membrane

Science.gov (United States)

Hardman, P.; Klement, B. J.; Spooner, B. S.

1993-01-01

Embryonic mouse salivary glands, pancreata, and kidneys were isolated from embryos of appropriate gestational age by microdissection, and were cultured on Biopore membrane either non-coated or coated with type I collagen or Matrigel. As expected, use of Biopore membrane allowed high quality photomicroscopy of the living organs. In all organs extensive mesenchymal spreading was observed in the presence of type I collagen or Matrigel. However, differences were noted in the effects of extracellular matrix (ECM) coatings on epithelial growth and morphogenesis: salivary glands were minimally affected, pancreas morphogenesis was adversely affected, and kidney growth and branching apparently was enhanced. It is suggested that these differences in behaviour reflect differences in the strength of interactions between the mesenchymal cells and their surrounding endogenous matrix, compared to the exogenous ECM macromolecules. This method will be useful for culture of these and other embryonic organs. In particular, culture of kidney rudiments on ECM-coated Biopore offers a great improvement over previously used methods which do not allow morphogenesis to be followed in vitro.

Plasma membrane proteomics of human embryonic stem cells and human embryonal carcinoma cells.

NARCIS (Netherlands)

Dormeyer, W.; van Hoof, D.; Braam, S.R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

2008-01-01

Human embryonic stem cells (hESCs) are of immense interest in regenerative medicine as they can self-renew indefinitely and can give rise to any adult cell type. Human embryonal carcinoma cells (hECCs) are the malignant counterparts of hESCs found in testis tumors. hESCs that have acquired

Contrasting Nephropathic Responses to Oral Administration of Extract of Cultured Penicillium polonicum in Rat and Primate

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John E. Fincham

2010-08-01

Full Text Available Liquid- or solid substrate-cultured Penicillium polonicum administered in feed to rats over several days evokes a histopathological response in kidney involving apoptosis and abnormal mitosis in proximal tubules. The amphoteric toxin is yet only partly characterized, but can be isolated from cultured sporulating biomass in a fraction that is soluble in water and ethanol, and exchangeable on either anion- or cation-exchange resins. After several weeks of treatment renal proximal tubule distortion became striking on account of karyocytomegaly, but even treatment for nearly two years remained asymptomatic. Extract from a batch of solid substrate fermentation of P. polonicum on shredded wheat was incorporated into feed for rats during four consecutive days, and also given as an aqueous solution by oral gavage to a vervet monkey daily for 10 days. Treatment was asymptomatic for both types of animal. Rat response was evident as the typical renal apoptosis and karyomegaly. In contrast there was no such response in the primate; and neither creatinine clearance nor any haematological characteristic or serum component concentration deviated from a control or from historical data for this primate. The contrast is discussed concerning other negative findings for P. polonicum in pigs and hamsters. Renal karyomegaly, as a common rat response to persistent exposure to ochratoxin A, is not known in humans suspected as being exposed to more than the usual trace amounts of dietary ochratoxin A. Therefore the present findings question assumptions that human response to ochratoxin A conforms to that in the rat.

Protection by free oxygen radical scavenging enzymes against salicylate-induced embryonic malformations in vitro.

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Karabulut, A K; Ulger, H; Pratten, M K

2000-08-01

Salicylates are among the oldest and most widely used drugs and are known to lead to foetal death, growth retardation and congenital abnormalities in experimental animals. In this study, the effects of acetyl salicylic acid (ASA), salicylic acid (SAL) and sodium salicylate (NaSAL) on early organogenesis and the interaction of these molecules with free radicals has been investigated. Postimplantation rat embryos were cultured in vitro from day 9.5 of gestation for 48 hr. ASA, SAL and NaSAL were added to whole rat serum at concentrations between 0.1 and 0.6 mg/ml. Also, the lowest effective concentration of ASA for all parameters (0.3 mg/ml) and the same concentration of NaSAL and SAL was added to the culture media in the presence of superoxide dismutase (SOD) (30 U/ml) or glutathione (0.5 micromol/ml). The growth and development of embryos was compared and each embryo was evaluated for the presence of any malformations. When compared to growth of control embryos, the salicylates decreased all growth and developmental parameters in a concentration-responsive manner. There was also a concentration-related increase in overall dysmorphology, including the incidence of haematoma in the yolk sac and neural system, open neural tube, abnormal tail torsion and the absence of fore limb bud. When SOD was added in the presence of ASA, growth and developmental parameters were improved and there was a significant decrease in the incidence of malformations. Addition of SOD also decreased the incidence of malformations in the presence of SAL, but did not effect the growth and developmental parameters of SAL and NaSAL. There was no significant difference between the embryos grown in the presence of these three molecules on the addition of glutathione. The effects of salicylates might involve free oxygen radicals by the non-enzymatic production of the highly teratogenic metabolites 2,3- and 2,5-dihydroxybenzoic acid. An enhanced production of these metabolites in embryonic tissues

A defined and xeno-free culture method enabling the establishment of clinical-grade human embryonic, induced pluripotent and adipose stem cells.

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Kristiina Rajala

2010-04-01

Full Text Available The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.Here, we report the development of a fully defined xeno-free medium (RegES, capable of supporting the expansion of human embryonic stem cells (hESC, induced pluripotent stem cells (iPSC and adipose stem cells (ASC. We describe the use of the xeno-free medium in the derivation and long-term (>80 passages culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS, while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific

Rats, cats, and elephants, but still no unicorn: induced pluripotent stem cells from new species.

Science.gov (United States)

Trounson, Alan

2009-01-09

Two independent studies in this issue of Cell Stem Cell (Liao et al., 2009; Li et al., 2009) derive rat induced pluripotent stem cells (iPSCs). In one report, the method used results in rat and human iPSCs that exhibit phenotypic traits similar to mouse embryonic stem cells.

A population of serumdeprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells

International Nuclear Information System (INIS)

Sauerzweig, Steven; Munsch, Thomas; Lessmann, Volkmar; Reymann, Klaus G.; Braun, Holger

2009-01-01

The bone marrow represents an easy accessible source of adult stem cells suitable for various cell based therapies. Several studies in recent years suggested the existence of pluripotent stem cells within bone marrow stem cells (BMSC) expressing marker proteins of both embryonic and tissue committed stem cells. These subpopulations were referred to as MAPC, MIAMI and VSEL-cells. Here we describe SD-BMSC (serumdeprivation-induced BMSC) which are induced as a distinct subpopulation after complete serumdeprivation. SD-BMSC are generated from small-sized nestin-positive BMSC (S-BMSC) organized as round-shaped cells in the top layer of BMSC-cultures. The generation of SD-BMSC is caused by a selective proliferation of S-BMSC and accompanied by changes in both morphology and gene expression. SD-BMSC up-regulate not only markers typical for neural stem cells like nestin and GFAP, but also proteins characteristic for embryonic cells like Oct4 and SOX2. We hypothesize, that SD-BMSC like MAPC, MIAMI and VSEL-cells represent derivatives from a single pluripotent stem cell fraction within BMSC exhibiting characteristics of embryonic and tissue committed stem cells. The complete removal of serum might offer a simple way to specifically enrich this fraction of pluripotent embryonic like stem cells in BMSC cultures

Depletion of Tcf3 and Lef1 maintains mouse embryonic stem cell self-renewal

OpenAIRE

Ye, Shoudong; Zhang, Tao; Tong, Chang; Zhou, Xingliang; He, Kan; Ban, Qian; Liu, Dahai; Ying, Qi-Long

2017-01-01

ABSTRACT Mouse and rat embryonic stem cell (ESC) self-renewal can be maintained by dual inhibition of glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase kinase (MEK). Inhibition of GSK3 promotes ESC self-renewal by abrogating T-cell factor 3 (TCF3)-mediated repression of the pluripotency network. How inhibition of MEK mediates ESC self-renewal, however, remains largely unknown. Here, we show that inhibition of MEK can significantly suppress lymphoid enhancer factor 1 (LEF1...

Enhanced expression of contractile endothelin ET(B) receptors in rat coronary artery after organ culture

DEFF Research Database (Denmark)

Johnsson, E.; Maddahi, A.; Wackenfors, A.

2008-01-01

. In cardiovascular disease and in organ culture in vitro, endothelin ET(B) receptors are up-regulated on smooth muscle cells. The objectives of the present study were to characterise the endothelin receptor-induced vasoconstriction and quantify the endothelin receptor mRNA levels and immunoreactivity in fresh...... and cultured rat coronary arteries. We demonstrate that endothelin-1 induces strong and equal concentration-dependent contractions in fresh and cultured segments from the left anterior descending coronary artery. Sarafotoxin 6c, an endothelin ET(B) receptor agonist, had negligible effect in fresh arteries...... but produced significant vasoconstriction after organ culture. The endothelin ET(B) receptor mRNA level and the receptor protein immunoreactivity were increased, whereas the level of endothelin ET(A) receptor mRNA was down-regulated but not its receptor protein immunoreactivity after organ culture...

In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes

International Nuclear Information System (INIS)

Burger, E.H.; Van der Meer, J.W.; van de Gevel, J.S.; Gribnau, J.C.; Thesingh, G.W.; van Furth, R.

1982-01-01

The origin of osteoclasts was studied in an in vitro model using organ cultures of periosteum-free embryonic mouse long-bone primordia, which were co-cultured with various cell populations. The bone rudiments were freed of their periosteum-perichondrium by collagenase treatment in a stage before cartilage erosion and osteoclast formation, and co-cultured for 7 d with either embryonic liver or mononuclear phagocytes from various sources. Light and electron microscopic examination of the cultures showed that mineralized matrix-resorbing osteoclasts developed only in bones co-cultured with embryonic liver or with cultured bone marrow mononuclear phagocytes but not when co-cultured with blood monocytes or resident or exudate peritoneal macrophages. Osteoclasts developed from the weakly adherent, but not from the strongly adherent cells of bone marrow cultures, whereas 1,000 rad irradiation destroyed the capacity of such cultures to form osteoclasts. In bone cultures to which no other cells were added, osteoclasts were virtually absent. Bone-resorbing activity of in vitro formed osteoclasts was demonstrated by 45 Ca release studies. These studies demonstrate that osteoclasts develop from cells present in cultures of proliferating mononuclear phagocytes and that, at least in our system, monocytes and macrophages are unable to form osteoclasts. The most likely candidates for osteoclast precursor cells seem to be monoblasts and promonocytes

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

International Nuclear Information System (INIS)

Varga, Nóra; Veréb, Zoltán; Rajnavölgyi, Éva; Német, Katalin; Uher, Ferenc; Sarkadi, Balázs; Apáti, Ágota

2011-01-01

Highlights: ► MSC like cells were derived from hESC by a simple and reproducible method. ► Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. ► MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

The Risk Evaluation of Tungsten Oxide Nanoparticles in Cultured Rat Liver Cells for Its Safe Applications in Nanotechnology

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Hasan Turkez

2014-08-01

Full Text Available Tungsten (VI oxide (WO3 nanoparticles (NPs are used for many industrial purposes in everyday life. However, their effects on human health have not been sufficiently evaluated. Therefore, the present study was designed to investigate the toxicity potentials of various concentrations (0 to 1000 ppm of WO3NPs (<100 nm particle size in cultured primary rat hepatocytes. The results of cell viability assay showed that the higher concentrations of dispersed WO3 NPs (300, 500 and 1000 ppm caused significant (p<0.05 decreases of cell viability. Also, dose dependent negative alterations were observed in oxidative status and antioxidant capacity levels after the application of WO3 in cultured rat primary hepatocytes. The results of genotoxicity tests revealed that these NPs did not cause significant increases of micronucleated hepatocytes (MNHEPs but increased 8-oxo-2-deoxyguanosine (8-OH-dG levels as compared to the control culture.

Nifedipine-activated Ca(2+) permeability in newborn rat cortical collecting duct cells in primary culture.

Science.gov (United States)

Valencia, L; Bidet, M; Martial, S; Sanchez, E; Melendez, E; Tauc, M; Poujeol, C; Martin, D; Namorado, M D; Reyes, J L; Poujeol, P

2001-05-01

To characterize Ca(2+) transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca(2+) concentration ([Ca(2+)](i)) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 microM) produced an increase in [Ca(2+)](i) from 87.6 +/- 3.3 nM to 389.9 +/- 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca(2+)](i) in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca(2+)](i). Experiments in the presence of EGTA showed that external Ca(2+) was required for the nifedipine effect, while lanthanum (20 microM), gadolinium (100 microM), and diltiazem (20 microM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K(+) channels were not involved in the nifedipine-induced [Ca(2+)](i) rise. H(2)O(2) also triggered [Ca(2+)](i) rise. However, nifedipine-induced [Ca(2+)](i) increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca(2+) transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca(2+) channel of capacitive type (either transient receptor potential or leak channel).

CaMKII and MEK1/2 inhibition time-dependently modify inflammatory signaling in rat cerebral arteries during organ culture

DEFF Research Database (Denmark)

Waldsee, Roya; Eftekhari, Sajedeh; Ahnstedt, Hilda

2014-01-01

MKII) II and extracellular signal-regulated kinase1/2 (ERK1/2) on inflammatory mediators in rat cerebral arteries using organ culture as a method for inducing ischemic-like vascular wall changes. METHODS: Rat basilar arteries were cultured in serum-free medium for 0, 3, 6 or 24 hours in the presence...... of phosphorylated c-Jun N-terminal kinase and p-p38, as evaluated by immunohistochemistry. KN93 affected the increase in caspase-3 mRNA expression only when given at the start of incubation, while U0126 had an inhibitory effect when given up to six hours later. Tumor necrosis factor receptor 1 was elevated after...

MDMA (Ecstasy) Decreases the Number of Neurons and Stem Cells in Embryonic Cortical Cultures

DEFF Research Database (Denmark)

Kindlundh-Högberg, Anna M S; Pickering, Chris; Wicher, Grzegorz

2010-01-01

Ecstasy, 3,4-methylenedioxymetamphetamine (MDMA), is a recreational drug used among adolescents, including young pregnant women. MDMA passes the placental barrier and may therefore influence fetal development. The aim was to investigate the direct effect of MDMA on cortical cells using dissociated...... CNS cortex of rat embryos, E17. The primary culture was exposed to a single dose of MDMA and collected 5 days later. MDMA caused a dramatic, dose-dependent (100 and 400 muM) decrease in nestin-positive stem cell density, as well as a significant reduction (400 muM) in NeuN-positive cells. By q......PCR, MDMA (200 muM) caused a significant decrease in mRNA expression of the 5HT3 receptor, dopamine D(1) receptor, and glutamate transporter EAAT2-1, as well as an increase in mRNA levels of the NMDA NR1 receptor subunit and the 5HT(1A) receptor. In conclusion, MDMA caused a marked reduction in stem cells...

Comparison of frailty of primary neurons, embryonic, and aging mouse cortical layers.

Science.gov (United States)

Fugistier, Patrick; Vallet, Philippe G; Leuba, Geneviève; Piotton, Françoise; Marin, Pascale; Bouras, Constantin; Savioz, Armand

2014-02-01

Superficial layers I to III of the human cerebral cortex are more vulnerable toward Aβ peptides than deep layers V to VI in aging. Three models of layers were used to investigate this pattern of frailty. First, primary neurons from E14 and E17 embryonic murine cortices, corresponding respectively to future deep and superficial layers, were treated either with Aβ(1-42), okadaic acid, or kainic acid. Second, whole E14 and E17 embryonic cortices, and third, in vitro separated deep and superficial layers of young and old C57BL/6J mice, were treated identically. We observed that E14 and E17 neurons in culture were prone to death after the Aβ and particularly the kainic acid treatment. This was also the case for the superficial layers of the aged cortex, but not for the embryonic, the young cortex, and the deep layers of the aged cortex. Thus, the aged superficial layers appeared to be preferentially vulnerable against Aβ and kainic acid. This pattern of vulnerability corresponds to enhanced accumulation of senile plaques in the superficial cortical layers with aging and Alzheimer's disease. Copyright © 2014 Elsevier Inc. All rights reserved.

Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK.

NARCIS (Netherlands)

Graichen, R.; Xu, X.; Braam, S.R.; Balakrishnan, T.; Norfiza, S.; Sieh, S.; Soo, S.Y.; Tham, S.C.; Mummery, C.L.; Colman, A.; Zweigerdt, R.; Davidson, B.P.

2008-01-01

Human embryonic stem cells (hESC) can differentiate to cardiomyocytes in vitro but with generally poor efficiency. Here, we describe a novel method for the efficient generation of cardiomyocytes from hESC in a scalable suspension culture process. Differentiation in serum-free medium conditioned by

Possible promotion of neuronal differentiation in fetal rat brain neural progenitor cells after sustained exposure to static magnetism.

Science.gov (United States)

Nakamichi, Noritaka; Ishioka, Yukichi; Hirai, Takao; Ozawa, Shusuke; Tachibana, Masaki; Nakamura, Nobuhiro; Takarada, Takeshi; Yoneda, Yukio

2009-08-15

We have previously shown significant potentiation of Ca(2+) influx mediated by N-methyl-D-aspartate receptors, along with decreased microtubules-associated protein-2 (MAP2) expression, in hippocampal neurons cultured under static magnetism without cell death. In this study, we investigated the effects of static magnetism on the functionality of neural progenitor cells endowed to proliferate for self-replication and differentiate into neuronal, astroglial, and oligodendroglial lineages. Neural progenitor cells were isolated from embryonic rat neocortex and hippocampus, followed by culture under static magnetism at 100 mT and subsequent determination of the number of cells immunoreactive for a marker protein of particular progeny lineages. Static magnetism not only significantly decreased proliferation of neural progenitor cells without affecting cell viability, but also promoted differentiation into cells immunoreactive for MAP2 with a concomitant decrease in that for an astroglial marker, irrespective of the presence of differentiation inducers. In neural progenitors cultured under static magnetism, a significant increase was seen in mRNA expression of several activator-type proneural genes, such as Mash1, Math1, and Math3, together with decreased mRNA expression of the repressor type Hes5. These results suggest that sustained static magnetism could suppress proliferation for self-renewal and facilitate differentiation into neurons through promoted expression of activator-type proneural genes by progenitor cells in fetal rat brain.

Synergistic toxicity of ethanol and MDMA towards primary cultured rat hepatocytes

International Nuclear Information System (INIS)

Pontes, Helena; Sousa, Carla; Silva, Renata; Fernandes, Eduarda; Carmo, Helena; Remiao, Fernando; Carvalho, Felix; Bastos, Maria Lourdes

2008-01-01

Ethanol is frequently consumed along with 3,4-methylenedioxymethamphetamine (MDMA; ecstasy). Since both compounds are hepatotoxic and are metabolized in the liver, an increased deleterious interaction resulting from the concomitant use of these two drugs seems plausible. Another important feature of MDMA-induced toxicity is hyperthermia, an effect known to be potentiated after continuous exposure to ethanol. Considering the potential deleterious interaction, the aim of the present study was to evaluate the hepatotoxic effects of ethanol and MDMA mixtures to primary cultured rat hepatocytes and to elucidate the mechanism(s) underlying this interaction. For this purpose, the toxicity induced by MDMA to primary cultured rat hepatocytes in absence or in presence of ethanol was evaluated, under normothermic (36.5 deg. C) and hyperthermic (40.5 deg. C) conditions. While MDMA and ethanol, by themselves, had discrete effects on the analysed parameters, which were slightly aggravated under hyperthermia, the simultaneous incubation of MDMA and ethanol for 24 h, resulted in high cell death ratios accompanied by a significant disturbance of cellular redox status and decreased energy levels. Evaluation of apoptotic/necrotic features provided clear evidences that the cell death occurs preferentially through a necrotic pathway. All the evaluated parameters were dramatically aggravated when cells were incubated under hyperthermia. In conclusion, co-exposure of hepatocytes to ethanol and MDMA definitely results in a synergism of the hepatotoxic effects, through a disruption of the cellular redox status and enhanced cell death by a necrotic pathway in a temperature-dependent extent

Mitochondrial activity assessed by cytofluorescence after in-vitro-irradiation of primary rat brain cultures

International Nuclear Information System (INIS)

Cervos-Navarro, J.; Hamdorf, G.

1993-01-01

Mitochondria play a key role in cell homeostasis and are the first cell organells affected by ionizing irradiation, as it was proved by previous electron microscopic investigations. In order to observe functional parameters of mitochondria after low-dose irradiation, primary rat brain cultures (prepared from 15-day-old rat fetuses) were irradiated from a 60 Co-source with 0.5 and 1 Gy at the age of 2 or 7 days in vitro (div). Cytofluorescence measurement was made by a Cytofluor trademark2350 using Rhodamine 123. This fluorescent dye is positively charged and accumulates specifically in the mitochondria of living cells without cytotoxic effect. Since its retention depends on the negative membrane potential as well as the proton gradient that exists across the inner mitochondrial membrane, Rhodamine 123 accumulation reflects the status of mitochondrial activity as a whole. After irradiation with 0.5 and 1 Gy on day 2 in culture there was a decrease in Rhodamine uptake in the irradiated cultures during the first week after the irradiation insult which reached minimum values after 3 days. Rhodamine uptake increased during the following period and finally reached the values of the control cultures. In the second experiment with irradiated cultures on day 7 and the same doses of 0.5 and 1 Gy the accumulation of Rhodamine decreased only initially then increased tremendously. After both doses values of Rhodamine-accumulation were higher than the control level. The results demonstrated that irradiation caused a change in mitochondrial activity depending on the time of irradiation. The dramatic increase over the control levels after irradiation on day 7 in vitro is attributed to the fact that at this time synapses have already developed. Deficiency of mitochondrial activity as well as hyperactivity and the consequent change in energy production may lead to changes in neuronal metabolism including an increase in production of free radicals

A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells

International Nuclear Information System (INIS)

Liu Yanxia; Song Zhihua; Zhao Yang; Qin Han; Cai Jun; Zhang Hong; Yu Tianxin; Jiang Siming; Wang Guangwen; Ding Mingxiao; Deng Hongkui

2006-01-01

Traditionally, undifferentiated human embryonic stem cells (hESCs) are maintained on mouse embryonic fibroblast (MEF) cells or on matrigel with an MEF-conditioned medium (CM), which hampers the clinical applications of hESCs due to the contamination by animal pathogens. Here we report a novel chemical-defined medium using DMEM/F12 supplemented with N2, B27, and basic fibroblast growth factor (bFGF) [termed NBF]. This medium can support prolonged self-renewal of hESCs. hESCs cultured in NBF maintain an undifferentiated state and normal karyotype, are able to form embryoid bodies in vitro, and differentiate into three germ layers and extraembryonic cells. Furthermore, we find that hESCs cultured in NBF possess a low apoptosis rate and a high proliferation rate compared with those cultured in MEF-CM. Our findings provide a novel, simplified chemical-defined culture medium suitable for further therapeutic applications and developmental studies of hESCs

How the embryonic chick brain twists

OpenAIRE

Chen, Zi; Guo, Qiaohang; Dai, Eric; Forsch, Nickolas; Taber, Larry A.

2016-01-01

During early development, the tubular embryonic chick brain undergoes a combination of progressive ventral bending and rightward torsion, one of the earliest organ-level left–right asymmetry events in development. Existing evidence suggests that bending is caused by differential growth, but the mechanism for the predominantly rightward torsion of the embryonic brain tube remains poorly understood. Here, we show through a combination of in vitro experiments, a physical model of the embryonic m...

Sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured rat caput epididymal epithelium.

Science.gov (United States)

Zuo, Wu-Lin; Li, Sheng; Huang, Jie-Hong; Yang, Deng-Liang; Zhang, Geng; Chen, Si-Liang; Ruan, Ye-Chun; Ye, Ke-Nan; Cheng, Christopher H K; Zhou, Wen-Liang

2011-01-01

The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na(+)/HCO(3)(-) cotransporter in the pH regulation in rat epididymis. Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit (KH) solution, the intracellular pH (pHi) recovery from NH(4)Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na(+)/H(+) exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO(3)(-) buffered would cause another pHi recovery. The pHi recovery in HCO(3)(-) buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), the inhibitor of HCO(3)(-) transporter or by removal of extracellular Na(+). The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH. The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.

Characterization of the onset of embryonic control and early development in the bovine embryo

International Nuclear Information System (INIS)

Barnes, F.L.

1988-01-01

Bovine embryos were used to determine if morphological and molecular features of early development are similar to in vivo recovered bovine embryos and to determine at what level early bovine development is regulated. Radiolabeling of IVP embryos and in vivo recovered embryos with 35 S-methionine for SDS-polyacrylamide gel electrophoresis reveals that these embryos are equivalent. Few differences in protein profiles are observed between 1- and early 4-cell embryos. A change in protein profiles begins at the mid 4-cell stage and continues into the 8-cell stage. Few differences in protein profiles are observed between 1- and early 4-cell embryos. A change in protein profiles begins at the mid 4-cell stage and continues into the 8-cell stage. Few differences in protein profiles are observed between late 8-cells and morulae. This transition is α-amanitin sensitive therefore due to de novo embryonic transcription. Embryonic transcription is partially responsible for terminating the post-transcriptionally regulated period of early bovine development. Argentophillic nucleolar organizing regions (Ag-NORs) indicate onset of nucleolar activation. Ag-NORs were absent in 2- and 4-cell IVP embryos and rarely occurred in 8-cell IVP embryos cultured in vitro. IVP 1- and 2-cell embryos cultured to blastocysts in sheep oviducts demonstrated Ag-NORs. Thus the lack of nucleolar activation of IVP embryos cultured in vitro is culture induced between the 2- and 8-cell stage

Disruption of murine mp29/Syf2/Ntc31 gene results in embryonic lethality with aberrant checkpoint response.

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Chia-Hsin Chen

Full Text Available Human p29 is a putative component of spliceosomes, but its role in pre-mRNA is elusive. By siRNA knockdown and stable overexpression, we demonstrated that human p29 is involved in DNA damage response and Fanconi anemia pathway in cultured cells. In this study, we generated p29 knockout mice (mp29(GT/GT using the mp29 gene trap embryonic stem cells to study the role of mp29 in DNA damage response in vivo. Interruption of mp29 at both alleles resulted in embryonic lethality. Embryonic abnormality occurred as early as E6.5 in mp29(GT/GT mice accompanied with decreased mRNA levels of α-tubulin and Chk1. The reduction of α-tubulin and Chk1 mRNAs is likely due to an impaired post-transcriptional event. An aberrant G2/M checkpoint was found in mp29 gene trap embryos when exposed to aphidicolin and UV light. This embryonic lethality was rescued by crossing with mp29 transgenic mice. Additionally, the knockdown of zfp29 in zebrafish resulted in embryonic death at 72 hours of development postfertilization (hpf. A lower level of acetylated α-tubulin was also observed in zfp29 morphants. Together, these results illustrate an indispensable role of mp29 in DNA checkpoint response during embryonic development.

Endocytosis of heat-denatured albumin by cultured rat Kupffer cells

International Nuclear Information System (INIS)

Brouwer, A.; Knook, D.L.

1982-01-01

Purified Kupffer cells were obtained by centrifugal elutriation of sinusoidal cells isolated by pronase treatment of the rat liver. The endocytosis of radioactively labeled heat-aggregated colloidal albumin (CA 125 I) was investigated in maintenance cultures of the purified Kupffer cells. The endocytic capacity of the cells was studied during 4 days of culture. Maximum uptake was observed after 24 hr of culture, with a gradual decline during the following days. When the uptake was measured after incubation with increasing concentrations of CA 125 I, a saturation effect was observed. This finding and the observed high rate of uptake are strong indications that receptor sites on the cell membrane are involved in the mechanism of endocytosis. The uptake of CA 125 I by Kupffer cells was inhibited by the metabolic inhibitors fluoride and antimycin A, indicating that endocytosis of CA 125 I is dependent on energy derived from both glycolysis and mitochondrial respiration. The mechanism of internalization may also require the action of microfilaments as well as intact microtubules, since both cytochalasin B and colchicine inhibited the uptake of CA 125 I. The intracellular degradation of CA 125 I by Kupffer cells was strongly inhibited by chloroquine but not by colchicine. The degradation of ingested CA 125 I occurred within the Kupffer cell lysosomes

Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells.

Science.gov (United States)

Soucie, Erinn L; Weng, Ziming; Geirsdóttir, Laufey; Molawi, Kaaweh; Maurizio, Julien; Fenouil, Romain; Mossadegh-Keller, Noushine; Gimenez, Gregory; VanHille, Laurent; Beniazza, Meryam; Favret, Jeremy; Berruyer, Carole; Perrin, Pierre; Hacohen, Nir; Andrau, J-C; Ferrier, Pierre; Dubreuil, Patrice; Sidow, Arend; Sieweke, Michael H

2016-02-12

Differentiated macrophages can self-renew in tissues and expand long term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network that controls self-renewal. Single-cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells. Copyright © 2016, American Association for the Advancement of Science.

Generation of Otic Sensory Neurons from Mouse Embryonic Stem Cells in 3D Culture

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Michael Perny

2017-12-01

Full Text Available The peripheral hearing process taking place in the cochlea mainly depends on two distinct sensory cell types: the mechanosensitive hair cells and the spiral ganglion neurons (SGNs. The first respond to the mechanical stimulation exerted by sound pressure waves on their hair bundles by releasing neurotransmitters and thereby activating the latter. Loss of these sensorineural cells is associated with permanent hearing loss. Stem cell-based approaches aiming at cell replacement or in vitro drug testing to identify potential ototoxic, otoprotective, or regenerative compounds have lately gained attention as putative therapeutic strategies for hearing loss. Nevertheless, they rely on efficient and reliable protocols for the in vitro generation of cochlear sensory cells for their implementation. To this end, we have developed a differentiation protocol based on organoid culture systems, which mimics the most important steps of in vivo otic development, robustly guiding mouse embryonic stem cells (mESCs toward otic sensory neurons (OSNs. The stepwise differentiation of mESCs toward ectoderm was initiated using a quick aggregation method in presence of Matrigel in serum-free conditions. Non-neural ectoderm was induced via activation of bone morphogenetic protein (BMP signaling and concomitant inhibition of transforming growth factor beta (TGFβ signaling to prevent mesendoderm induction. Preplacodal and otic placode ectoderm was further induced by inhibition of BMP signaling and addition of fibroblast growth factor 2 (FGF2. Delamination and differentiation of SGNs was initiated by plating of the organoids on a 2D Matrigel-coated substrate. Supplementation with brain-derived neurotrophic factor (BDNF and neurotrophin-3 (NT-3 was used for further maturation until 15 days of in vitro differentiation. A large population of neurons with a clear bipolar morphology and functional excitability was derived from these cultures. Immunostaining and gene expression

Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes

International Nuclear Information System (INIS)

Junker, L.H.; Davis, R.A.

1989-01-01

The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of [14C]cholesterol from [2-14C]acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of [14C]cholesterol from [2-14C]acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis

Role of cyclic GMP in cells with the properties of smooth muscle cultured from the rat myometrium

International Nuclear Information System (INIS)

Krall, J.F.; Morin, A.

1986-01-01

Cells growing in culture with previously described properties of rat uterine smooth muscle accumulated 45 Ca 2+ from the medium. Ca 2+ uptake by these cells was stimulated by the addition to the medium of 8-bromo-cGMP but not by 8-bromo-cAMP. Ca 2+ uptake was also stimulated by carbachol and by the nitro-vasodilator nitroprusside. Although cholinergic agonists have been shown previously to stimulate contraction but not cGMP synthesis in the rat myometrium, both carbachol and nitroprusside stimulated cGMP production by the cultured cells. These results suggested the cells had cholinergic receptor-medicated functions that reflected some neurotransmitter-sensitive properties of uterine smooth muscle in situ. When determined by a specific radioligand binding assay, subcellular fractions of the cultured cells bound muscarinic cholinergic agonists and antagonists with affinities expected of the muscarinic receptor. The cells were also sensitive to the β-adrenergic catecholamine agonist isoproterenol, which stimulated cAMP production but not Ca 2+ uptake. Carbachol failed to inhibit isoproterenol-dependent cAMP production, which is an important property of the cholinergic receptor in uterine smooth muscle in situ. These results suggest some but not all acetylcholine-sensitive properties of uterine smooth muscle may be retained in cell culture

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

Energy Technology Data Exchange (ETDEWEB)

Varga, Nora [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Vereb, Zoltan; Rajnavoelgyi, Eva [Department of Immunology, Medical and Health Science Centre, University of Debrecen, Debrecen (Hungary); Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Apati, Agota, E-mail: apati@kkk.org.hu [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary)

2011-10-28

Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector.

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Claudia Merkl

Full Text Available Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4 into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.

Ethanol affects network activity in cultured rat hippocampus: mediation by potassium channels.

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Eduard Korkotian

Full Text Available The effects of ethanol on neuronal network activity were studied in dissociated cultures of rat hippocampus. Exposure to low (0.25-0.5% ethanol concentrations caused an increase in synchronized network spikes, and a decrease in the duration of individual spikes. Ethanol also caused an increase in rate of miniature spontaneous excitatory postsynaptic currents. Higher concentrations of ethanol eliminated network spikes. These effects were reversible upon wash. The effects of the high, but not the low ethanol were blocked by the GABA antagonist bicuculline. The enhancing action of low ethanol was blocked by apamin, an SK potassium channel antagonist, and mimicked by 1-EBIO, an SK channel opener. It is proposed that in cultured hippocampal networks low concentration of ethanol is associated with SK channel activity, rather than the GABAergic receptor.

Permanently Hypoxic Cell Culture Yields Rat Bone Marrow Mesenchymal Cells with Higher Therapeutic Potential in the Treatment of Chronic Myocardial Infarction

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Yihua Liu

2017-11-01

Full Text Available Background: The mismatch between traditional in vitro cell culture conditions and targeted chronic hypoxic myocardial tissue could potentially hamper the therapeutic effects of implanted bone marrow mesenchymal stem cells (BMSCs. This study sought to address (i the extent of change to BMSC biological characteristics in different in vitro culture conditions and (ii the effectiveness of permanent hypoxic culture for cell therapy in treating chronic myocardial infarction (MI in rats. Methods: rat BMSCs were harvested and cultured in normoxic (21% O2, n=27 or hypoxic conditions (5% O2, n=27 until Passage 4 (P4. Cell growth tests, flow cytometry, and Bio-Plex assays were conducted to explore variations in the cell proliferation, phenotype, and cytokine expression, respectively. In the in vivo set-up, P3-BMSCs cultured in normoxia (n=6 or hypoxia (n=6 were intramyocardially injected into rat hearts that had previously experienced 1-month-old MI. The impact of cell therapy on cardiac segmental viability and hemodynamic performance was assessed 1 month later by 2-Deoxy-2[18F]fluoro-D-glucose (18F-FDG positron emission tomography (PET imaging and pressure-volume catheter, respectively. Additional histomorphological examinations were conducted to evaluate inflammation, fibrosis, and neovascularization. Results: Hypoxic preconditioning significantly enhanced rat BMSC clonogenic potential and proliferation without altering the multipotency. Different profiles of inflammatory, fibrotic, and angiogenic cytokine secretion were also documented, with a marked correlation observed between in vitro and in vivo proangiogenic cytokine expression and tissue neovessels. Hypoxic-preconditioned cells presented a beneficial effect on the myocardial viability of infarct segments and intrinsic contractility. Conclusion: Hypoxic-preconditioned BMSCs were able to benefit myocardial perfusion and contractility, probably by modulating the inflammation and promoting

Permanently Hypoxic Cell Culture Yields Rat Bone Marrow Mesenchymal Cells with Higher Therapeutic Potential in the Treatment of Chronic Myocardial Infarction.

Science.gov (United States)

Liu, Yihua; Yang, Xiaoxi; Maureira, Pablo; Falanga, Aude; Marie, Vanessa; Gauchotte, Guillaume; Poussier, Sylvain; Groubatch, Frederique; Marie, Pierre-Yves; Tran, Nguyen

2017-01-01

The mismatch between traditional in vitro cell culture conditions and targeted chronic hypoxic myocardial tissue could potentially hamper the therapeutic effects of implanted bone marrow mesenchymal stem cells (BMSCs). This study sought to address (i) the extent of change to BMSC biological characteristics in different in vitro culture conditions and (ii) the effectiveness of permanent hypoxic culture for cell therapy in treating chronic myocardial infarction (MI) in rats. rat BMSCs were harvested and cultured in normoxic (21% O2, n=27) or hypoxic conditions (5% O2, n=27) until Passage 4 (P4). Cell growth tests, flow cytometry, and Bio-Plex assays were conducted to explore variations in the cell proliferation, phenotype, and cytokine expression, respectively. In the in vivo set-up, P3-BMSCs cultured in normoxia (n=6) or hypoxia (n=6) were intramyocardially injected into rat hearts that had previously experienced 1-month-old MI. The impact of cell therapy on cardiac segmental viability and hemodynamic performance was assessed 1 month later by 2-Deoxy-2[18F]fluoro-D-glucose (18F-FDG) positron emission tomography (PET) imaging and pressure-volume catheter, respectively. Additional histomorphological examinations were conducted to evaluate inflammation, fibrosis, and neovascularization. Hypoxic preconditioning significantly enhanced rat BMSC clonogenic potential and proliferation without altering the multipotency. Different profiles of inflammatory, fibrotic, and angiogenic cytokine secretion were also documented, with a marked correlation observed between in vitro and in vivo proangiogenic cytokine expression and tissue neovessels. Hypoxic-preconditioned cells presented a beneficial effect on the myocardial viability of infarct segments and intrinsic contractility. Hypoxic-preconditioned BMSCs were able to benefit myocardial perfusion and contractility, probably by modulating the inflammation and promoting angiogenesis. © 2017 The Author(s). Published by S. Karger AG

Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21) and Nsg-2 (P19).

Science.gov (United States)

Digilio, Laura; Yap, Chan Choo; Winckler, Bettina

2015-01-01

The brain consists of many distinct neuronal cell types, but which cell types are present in widely used primary cultures of embryonic rodent brain is often not known. We characterized how abundantly four cell type markers (Ctip2, Satb2, Prox1, GAD65) were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21) and Nsg-2 (P19) are ubiquitously expressed in all types of cultured neurons. We found that cultured neurons stably maintain cell type identities that are reflective of cell types in vivo. This includes neurons maintaining simultaneous expression of two transcription factors, such as Ctip2+/Satb2+ or Prox1+/Ctip2+ double-positive cells, which have also been described in vivo. Secondly, we established the superior efficiency of CAG promoters for both Lipofectamine-mediated transfection as well as for electroporation. Thirdly, we discovered that Nsg-1 and Nsg-2 were not expressed equally in all neurons: whereas high levels of both Nsg-1 and Nsg-2 were found in Satb2-, Ctip2-, and GAD65-positive neurons, Prox1-positive neurons in hippocampal cultures expressed low levels of both. Our findings thus highlight the importance of identifying neuronal cell types for doing cell biology in cultured neurons: Keeping track of neuronal cell type might uncover effects in assays that might otherwise be masked by the mixture of responsive and non-responsive neurons in the dish.

Infection in a rat model reactivates attenuated virulence after long-term axenic culture of Acanthamoeba spp

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Carolina De Marco Verissimo

2013-11-01

Full Text Available Prolonged culturing of many microorganisms leads to the loss of virulence and a reduction of their infective capacity. However, little is known about the changes in the pathogenic strains of Acanthamoeba after long culture periods. Our study evaluated the effect of prolonged culturing on the invasiveness of different isolates of Acanthamoeba in an in vivo rat model. ATCC strains of Acanthamoeba, isolates from the environment and clinical cases were evaluated. The in vivo model was effective in establishing the infection and differentiating the pathogenicity of the isolates and re-isolates. The amoebae cultured in the laboratory for long periods were less virulent than those that were recently isolated, confirming the importance of passing Acanthamoeba strains in animal models.

Importance of a diffusion-dominant small volume to activate cell-secreted soluble factor signaling in embryonic stem cell culture in microbioreactors: a mathematical model based study.

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Chowdhury, Mohammad Mahfuz; Fujii, Teruo; Sakai, Yasuyuki

2013-07-01

In our previous studies, we observed that cell-secreted BMP4 had a prominent influence on mouse embryonic stem cell (mESC) behaviors in a membrane-based two-chambered microbioreactor (MB), but not in a macro-scale culture (6-well plate/6WP). In this study, we investigated how the physical aspects of these cultures regulated BMP4 signaling by developing mathematical models of the cultures. The models estimated signaling activity in the cultures by considering size of the undifferentiated mESC colonies and their growth, diffusion of BMP4, and BMP4 trafficking process in the colonies. The models successfully depicted measured profile of BMP4 concentration in the culture medium which was two times higher in the MB than that in the 6WP during 5-day culture. The models estimated that, owing to the small volume and the membrane, cells were exposed to a higher BMP4 concentration in the top chamber of the MB than that in the 6WP culture. The higher concentration of BMP4 induced a higher concentration of BMP4-bound receptor in the colony in the MB than in the 6WP, thereby leading to the higher activation of BMP4 signaling in the MB. The models also predicted that the size of the MB, but not that of the 6WP, was suitable for maximizing BMP4 accumulation and upregulating its signaling. This study will be helpful in analyzing culture systems, designing microfluidic devices for controlling ESC or other cell behavior. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Effect of oxygen deprivation on metabolism of arachidonic acid by cultures of rat heart cells

International Nuclear Information System (INIS)

Freyss-Beguin, M.; Millanvoye-van Brussel, E.; Duval, D.

1989-01-01

To investigate the mechanisms responsible for the impairment of phospholipid metabolism observed in ischemic cells, we have studied the effect of conditions simulating ischemia on the metabolism of arachidonic acid (AA) by muscle (M-) and nonmuscle (F-) cells isolated from newborn rat hearts and cultured separately. In muscle cells, oxygen deprivation induces a significant stimulation of the release of [ 14 C]AA from prelabeled cells associated with a preferential redistribution of [ 14 C]AA into cell triglycerides but not formation of radioactive prostaglandins. Moreover, the fatty acid content of phospholipids, as measured by capillary gas chromatography, appears markedly reduced in ischemic myocardial cells. This fact may be related to phospholipase stimulation during ischemia as suggested by the antagonistic effect of mepacrine or p-bromophenacyl bromide. In contrast, oxygen deprivation failed to induce any significant alteration of AA metabolism in fibroblast-like heart cells. Our results indicate that these cultures of newborn rat heart cells, which exhibit many of the features observed in intact organ during ischemia, may represent a useful experimental model to investigate the pharmacological control of the membrane phospholipid turnover

Adenosine formation in contracting primary rat skeletal muscle cells and endothelial cells in culture

DEFF Research Database (Denmark)

Hellsten, Ylva; Frandsen, Ulrik

1997-01-01

1. The present study examined the capacity for adenosine formation, uptake and metabolism in contracting primary rat muscle cells and in microvascular endothelial cells in culture. 2. Strong and moderate electrical simulation of skeletal muscle cells led to a significantly greater increase....... 3. Addition of microvascular endothelial cells to the cultured skeletal muscle cells enhanced the contraction-induced accumulation of extracellular adenosine (P Skeletal muscle cells were...... in the extracellular adenosine concentration (421 +/- 91 and 235 +/- 30 nmol (g protein)-1, respectively; P muscle cells (161 +/- 20 nmol (g protein)-1). The ATP concentration was lower (18%; P contracted, but not in the moderately contracted muscle cells...

Periodic harvesting of embryonic stem cells from a hollow-fiber membrane based four-compartment bioreactor.

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Knöspel, Fanny; Freyer, Nora; Stecklum, Maria; Gerlach, Jörg C; Zeilinger, Katrin

2016-01-01

Different types of stem cells have been investigated for applications in drug screening and toxicity testing. In order to provide sufficient numbers of cells for such in vitro applications a scale-up of stem cell culture is necessary. Bioreactors for dynamic three-dimensional (3D) culture of growing cells offer the option for culturing large amounts of stem cells at high densities in a closed system. We describe a method for periodic harvesting of pluripotent stem cells (PSC) during expansion in a perfused 3D hollow-fiber membrane bioreactor, using mouse embryonic stem cells (mESC) as a model cell line. A number of 100 × 10(6) mESC were seeded in bioreactors in the presence of mouse embryonic fibroblasts (MEF) as feeder cells. Over a cultivation interval of nine days cells were harvested by trypsin perfusion and mechanical agitation every second to third culture day. A mean of 380 × 10(6) mESC could be removed with every harvest. Subsequent to harvesting, cells continued growing in the bioreactor, as determined by increasing glucose consumption and lactate production. Immunocytochemical staining and mRNA expression analysis of markers for pluripotency and the three germ layers showed a similar expression of most markers in the harvested cells and in mESC control cultures. In conclusion, successful expansion and harvesting of viable mESC from bioreactor cultures with preservation of sterility was shown. The present study is the first one showing the feasibility of periodic harvesting of adherent cells from a continuously perfused four-compartment bioreactor including further cultivation of remaining cells. © 2015 American Institute of Chemical Engineers.

Histone h1 depletion impairs embryonic stem cell differentiation.

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Zhang, Yunzhe; Cooke, Marissa; Panjwani, Shiraj; Cao, Kaixiang; Krauth, Beth; Ho, Po-Yi; Medrzycki, Magdalena; Berhe, Dawit T; Pan, Chenyi; McDevitt, Todd C; Fan, Yuhong

2012-01-01

Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes.

Rat intermediate lobe in culture: a histological and biochemical characterization.

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Chronwall, B M; Bishop, J F; Gehlert, D R

1988-01-01

The histology, immunohistochemistry, peptide synthesis and secretion as well as proliferation rate of rat intermediate lobe (IL) were studied in primary cultures. The cultures contained two populations of cells: melanotrophs either organized in free floating lobules or in lobules which attached to the dishes and formed a monolayer. Both populations retained their in vivo morphology: polyhedral cells with smooth, ovoid nuclei and a large number of cytoplasmic secretory coated vesicles, a well developed Golgi apparatus, abundant mitochondria and extensive areas of rough endoplasmic reticulum. The melanotrophs stained with varying intensity for alpha-MSH and in situ hybridization showed the presence of pro-opiomelanocortin (POMC) mRNA. 35S-methionine incorporation combined with 2-D gel electrophoresis demonstrated POMC peptide synthesis and radioimmunoassay confirmed its secretion into the medium. 3H-thymidine uptake in the attached melanotrophs was considerably higher than that in the free-floating melanotrophs, demonstrating the dependency of proliferation rate on the cytoarchitecture of the explant. The retention of melanotroph morphology, biosynthetic and proliferative capacity in vitro affords a valid model system for studying POMC gene expression.

Primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue.

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Tamashiro, Tami T; Dalgard, Clifton Lee; Byrnes, Kimberly R

2012-08-15

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted. Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study. The principle and protocol of this methodology have been described in the literature. Additionally, alternate methodologies to isolate primary microglia are well described. Homogenized brain tissue may be separated

Chronic 14-day exposure to insecticides or methylmercury modulates neuronal activity in primary rat cortical cultures

NARCIS (Netherlands)

Dingemans, Milou; Schütte, Marijke G; Wiersma, Daphne M M; de Groot, Aart; van Kleef, Gina; Wijnolts, Fiona; Westerink, Remco

2016-01-01

There is an increasing demand for in vitro test systems to detect neurotoxicity for use in chemical risk assessment. In this study, we evaluated the applicability of rat primary cortical cultures grown on multi-well micro-electrode arrays (mwMEAs) to detect effects of chronic 14-day exposure to

Specific receptor for endothelin in cultured rat cardiocytes

International Nuclear Information System (INIS)

Hirata, Y.; Fukuda, Y.; Yoshimi, H.; Emori, T.; Shichiri, M.; Marumo, F.

1989-01-01

Specific binding sites for the endothelium-derived vasoconstrictor endothelin (ET) and its effect on cytosolic free Ca2+ concentrations [( Ca2+]i) were studied in a primary culture of cardiocytes from neonatal rats. Binding studies using 125 I-labeled-porcine ET as a radioligand revealed the presence of a single class of high-affinity binding sites for ET in cardiocytes with an apparent Kd of 6-9 x 10(-10) M and a Bmax of 50,000-80,000 sites/cell. Neither various vasoconstrictors nor Ca2+-channel blockers affected the binding. Pretreatment with ET substantially reduced the total number of ET receptors without changing their affinity. ET dose-dependently increased [Ca2+]i in fura-2-loaded cardiocytes. These data indicate that cardiocytes have specific ET receptors that are controlled by a down-regulation mechanism, and that ET induces a receptor-mediated increase in [Ca2+]i in cardiocytes

Endogenous hydrogen peroxide production in the epithelium of the developing embryonic lens.

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Basu, Subhasree; Rajakaruna, Suren; Dickinson, Bryan C; Chang, Christopher J; Menko, A Sue

2014-01-01

Hydrogen peroxide (H2O2) is an endogenously produced reactive oxygen species (ROS) present in a variety of mammalian systems. This particular ROS can play dichotomous roles, being beneficial in some cases and deleterious in others, which reflects the level and location of H2O2 production. While much is known about the redox regulation of ROS by antioxidant and repair systems in the lens, little is known about the endogenous production of H2O2 in embryonic lens tissue or the physiologic relevance of endogenous H2O2 to lens development. This gap in knowledge exists primarily from a lack of reagents that can specifically detect endogenous H2O2 in the intact lens. Here, using a recently developed chemoselective fluorescent boronate probe, peroxyfluor-6 acetoxymethyl ester (PF6-AM), which selectively detects H2O2 over related ROS, we examined the endogenous H2O2 signals in the embryonic lens. Embryonic day 10 chick whole lenses in ex vivo organ culture and lens epithelial cells in primary culture were loaded with the H2O2 probe PF6-AM. To determine the relationship between localization of mitochondria with active membrane potential and the region of H2O2 production in the lens, cells were exposed to the mitochondrial probe MitoTracker Red CMXRos together with PF6-AM. Diphenyleneiodonium (DPI), a flavin inhibitor that blocks generation of intracellular ROS production, was used to confirm that the signal from PF6-AM was due to endogenous ROS production. All imaging was performed by live confocal microscopy. PF6-AM detected endogenous H2O2 in lens epithelial cells in whole lenses in ex vivo culture and in lens epithelial cells grown in primary culture. No endogenous H2O2 signal could be detected in differentiating lens fiber cells with this probe. Treatment with DPI markedly attenuated the fluorescence signal from the peroxide-specific probe PF6-AM in the lens epithelium, suggesting that basal generation of ROS occurs in this region. The lens epithelial cells producing an

Embryonic Stem Cells-loaded Gelatin Microcryogels Slow Progression of Chronic Kidney Disease

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Geng, Xiao-Dong; Zheng, Wei; Wu, Cong-Mei; Wang, Shu-Qiang; Hong, Quan; Cai, Guang-Yan; Chen, Xiang-Mei; Wu, Di

2016-01-01

Background: Chronic kidney disease (CKD) has become a public health problem. New interventions to slow or prevent disease progression are urgently needed. In this setting, cell therapies associated with regenerative effects are attracting increasing interest. We evaluated the effect of embryonic stem cells (ESCs) on the progression of CKD. Methods: Adult male Sprague–Dawley rats were subjected to 5/6 nephrectomy. We used pedicled greater omentum flaps packing ESC-loaded gelatin microcryogels (GMs) on the 5/6 nephrectomized kidney. The viability of ESCs within the GMs was detected using in vitro two-photon fluorescence confocal imaging. Rats were sacrificed after 12 weeks. Renal injury was evaluated using serum creatinine, urea nitrogen, 24 h protein, renal pathology, and tubular injury score results. Structural damage was evaluated by periodic acid-Schiff and Masson trichrome staining. Results: In vitro, ESCs could be automatically loaded into the GMs. Uniform cell distribution, good cell attachment, and viability were achieved from day 1 to 7 in vitro. After 12 weeks, in the pedicled greater omentum flaps packing ESC-loaded GMs on 5/6 nephrectomized rats group, the plasma urea nitrogen levels were 26% lower than in the right nephrectomy group, glomerulosclerosis index was 62% lower and tubular injury index was 40% lower than in the 5/6 nephrectomized rats group without GMs. Conclusions: In a rat model of established CKD, we demonstrated that the pedicled greater omentum flaps packing ESC-loaded GMs on the 5/6 nephrectomized kidney have a long-lasting therapeutic rescue function, as shown by the decreased progression of CKD and reduced glomerular injury. PMID:26879011

Atorvastatin prevents Aβ oligomer-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting Tau cleavage

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Sui, Hai-juan; Zhang, Ling-ling; Liu, Zhou; Jin, Ying

2015-01-01

Aim: The proteolytic cleavage of Tau is involved in Aβ-induced neuronal dysfunction and cell death. In this study, we investigated whether atorvastatin could prevent Tau cleavage and hence prevent Aβ1–42 oligomer (AβO)-induced neurotoxicity in cultured cortical neurons. Methods: Cultured rat hippocampal neurons were incubated in the presence of AβOs (1.25 μmol/L) with or without atorvastatin pretreatment. ATP content and LDH in the culture medium were measured to assess the neuronal viability. Caspase-3/7 and calpain protease activities were detected. The levels of phospho-Akt, phospho-Erk1/2, phospho-GSK3β, p35 and Tau proteins were measured using Western blotting. Results: Treatment of the neurons with AβO significantly decreased the neuronal viability, induced rapid activation of calpain and caspase-3/7 proteases, accompanied by Tau degradation and relatively stable fragments generated in the neurons. AβO also suppressed Akt and Erk1/2 kinase activity, while increased GSK3β and Cdk5 activity in the neurons. Pretreatment with atorvastatin (0.5, 1, 2.5 μmol/L) dose-dependently inhibited AβO-induced activation of calpain and caspase-3/7 proteases, and effectively diminished the generation of Tau fragments, attenuated synaptic damage and increased neuronal survival. Atorvastatin pretreatment also prevented AβO-induced decreases in Akt and Erk1/2 kinase activity and the increases in GSK3β and Cdk5 kinase activity. Conclusion: Atorvastatin prevents AβO-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting calpain- and caspase-mediated Tau cleavage. PMID:25891085

Sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured rat caput epididymal epithelium.

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Wu-Lin Zuo

Full Text Available The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na(+/HCO(3(- cotransporter in the pH regulation in rat epididymis.Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit (KH solution, the intracellular pH (pHi recovery from NH(4Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na(+/H(+ exchanger (NHE. Immediately changing of the KH solution from HEPES buffered to HCO(3(- buffered would cause another pHi recovery. The pHi recovery in HCO(3(- buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, the inhibitor of HCO(3(- transporter or by removal of extracellular Na(+. The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH.The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.

Oocyte exposure to ZnO nanoparticles inhibits early embryonic development through the γ-H2AX and NF-κB signaling pathways.

Science.gov (United States)

Liu, Jing; Zhao, Yong; Ge, Wei; Zhang, Pengfei; Liu, Xinqi; Zhang, Weidong; Hao, Yanan; Yu, Shuai; Li, Lan; Chu, Meiqiang; Min, Lingjiang; Zhang, Hongfu; Shen, Wei

2017-06-27

The impacts of zinc oxide nanoparticles on embryonic development following oocyte stage exposure are unknown and the underlying mechanisms are sparsely understood. In the current investigation, intact nanoparticles were detected in ovarian tissue in vivo and cultured cells in vitro under zinc oxide nanoparticles treatment. Zinc oxide nanoparticles exposure during the oocyte stage inhibited embryonic development. Notably, in vitro culture data closely matched in vivo embryonic data, in that the impairments caused by Zinc oxide nanoparticles treatment passed through cell generations; and both gamma-H2AX and NF-kappaB pathways were involved in zinc oxide nanoparticles caused embryo-toxicity. Copper oxide and silicon dioxide nanoparticles have been used to confirm that particles are important for the toxicity of zinc oxide nanoparticles. The toxic effects of zinc oxide nanoparticles emanate from both intact nanoparticles and Zn2+. Our investigation along with others suggests that zinc oxide nanoparticles are toxic to the female reproductive system [ovaries (oocytes)] and subsequently embryo-toxic and that precaution should be taken regarding human exposure to their everyday use.

Non-specific interference of certain components of tissue culture media with the radioimmunoassay of rat alpha-foetoprotein

International Nuclear Information System (INIS)

Dambuyant, C.; Sizaret, Ph.

1975-01-01

Interferences of 'Williams' tissue culture medium used for cultivating rat hepatocytes upon rat alpha-foetoprotein (AFP) radioimmunoassay have been investigated. They are not due to foetal calf serum proteins which are added as growth factor and can be abolished by dialysis which appears to be necessary for the distinction between AFP non-producer and low-producer cell lines. Of the three major groups of non-mineral components examined, amino acid solution played a major role. When individual amino acids were examined using the double antibody technique, arginine was found to interfere predominantly; its dose-response curve was parallel to that of rat AFP which confirmed that an immunological identity between two substances cannot be established on the basis of parallelism as the only criterion

Female parthenogenetic apomixis and androsporogenetic parthenogenesis in embryonal cells of Araucaria angustifolia: interpolation of progenesis and asexual heterospory in an artificial sporangium.

Science.gov (United States)

Durzan, Don J

2012-09-01

Cell fate, development timing and occurrence of reproductive versus apomictic development in gymnosperms are shown to be influenced by culture conditions in vitro. In this study, female parthenogenetic apomixis (fPA), androsporogenetic parthenogenesis (mAP) and progenesis were demonstrated using embryonal initials of Araucaria angustifolia in scaled-up cell suspensions passing through a single-cell bottleneck in darkness and in an artificial sporangium (AS). Expression was based on defined nutrition, hormones and feedforward-adaptive feedback process controls at 23-25 °C and in darkness. In fPA, the nucleus of an embryonal initial undergoes endomitosis and amitosis, forming a diploid egg-equivalent and an apoptotic ventral canal nucleus in a transdifferentiated archegonial tube. Discharge of egg-equivalent cells as parthenospores and their dispersal into the aqueous culture medium were followed by free-nuclear conifer-type proembryogenesis. This replaced the plesiomorphic and central features of proembryogenesis in Araucariaceae. Protoplasmic fusions of embryonal initials were used to reconstruct heterokaryotic expressions of fPA in multiwell plates. In mAP, restitutional meiosis (automixis) was responsible for androsporogenesis and the discharge of monads, dyads, tetrads and polyads. In a display of progenesis, reproductive development was brought to an earlier ontogenetic stage and expressed by embryonal initials. Colchicine increased polyploidy, but androspore formation became aberrant and fragmented. Aberrant automixis led to the formation of chromosomal bouquets, which contributed to genomic silencing in embryonal initials, cytomixis and the formation of pycnotic micronucleated cells. Dispersal of female and male parthenospores displayed heteromorphic asexual heterospory in an aqueous environment.

Culturated rat cerebral cortex explants and their application in the study of SPECT scan radiopharaceuticals

International Nuclear Information System (INIS)

Jong, B.M. de.

1989-01-01

In this thesis mechanics that result in the distinct localization of radiopharmaceuticals within the brain have been investigated. In order to 'get more insight' in uptake and binding of radiopharmaceuticals bu brain tissue, use has been made of the tissue culture technique. Tissue culture privides the opportunity of doing experiments with brain tissue under stable conditions, in the absence of a blood-brain barrier, and without interference by cerebral blood flow. The present thesis is presented in two sections. The first part focusses on longterm culture of 'organotypic' cerebral neocortex tissue, obtained from neonatal rat brain and explanted into a chemically defined medium. Procedures were developed which enabled culturing of this tissue without the occurence of central necrosis and with the preservation of a characteristic histiotypic organization. Morphological characteristics of the cultures were described and measured at various ages in vitro. In the second part, the cultures were used to study mechanisms that might contribute to the tissue uptake of radiopharmaceuticals which are in clinical use for SPECT brain imaging. (author). 369 refs.; 50 figs.; 13 tabs

Distribution of phospholipase C isozymes in various rat tissues and cultured cells

International Nuclear Information System (INIS)

Suh, P.G.; Ryu, S.H.; Choi, W.C.; Lee, K.Y.; Rhee, S.G.

1987-01-01

Monoclonal antibodies prepared against PLC-I or PLC-II enzyme did not cross-react with the other. Using a pair of antibodies which recognizes 2 different antigenic sites on the same molecule, radioimmunoassays were developed for the quantitation of PLC-I and PLC-II in homogenates of various tissues and cultured cells, prepared by homogenization in a 2 M KCl buffer. The contents of PLC enzymes were measured in 19 rat tissues, in human platelets and in 17 cultured cells. Results indicate that the concentration of PLC-I and PLC-II is very high in brain, PLC-I is localized mainly in brain and partly in seminal vesicles, PLC-II is found in most tissues and cells. PLC-I is highly localized even in brain: 5 different neuroblastoma did not contain PLC-I while 2 glioma and 1 astrocytoma contained significant amounts

The endozepine ODN stimulates [3H]thymidine incorporation in cultured rat astrocytes

International Nuclear Information System (INIS)

Gandolfo, P.; Patte, C.; Thoumas, J.L.; Leprince, J.; Vaudry, H.; Tonon, M.C.

1999-01-01

High concentrations of diazepam-binding inhibitor (DBI) mRNA have been detected in astrocytoma, suggesting that DBI-derived peptides may play a role in glial cell proliferation. In the present study, we have investigated the effect of a processing product of DBI, the octadecaneuropeptide ODN, on DNA synthesis in cultured rat astrocytes. At very low concentrations (10 -14 to 10 -11 M), ODN caused a dose-dependent increase of [ 3 H]thymidine incorporation. At higher doses (10 -10 to 10 -5 M), the effect of ODN gradually declined. The central-type benzodiazepine receptor antagonist flumazenil (10 -6 M) completely suppressed the stimulatory action of ODN whereas the peripheral-type benzodiazepine receptor ligand, PK11195 (10 -6 M) had no effect. The ODN-induced stimulation of [ 3 H]thymidine incorporation was mimicked by methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM). The GABA A receptor antagonist bicuculline (10 -4 M) suppressed the effect of both ODN and DMCM on DNA synthesis. Exposure of cultured astrocytes to the specific GABA A agonist 3APS (10 -10 to 10 -4 M) also induced a dose-related increase of [ 3 H]thymidine incorporation. The present study indicates that ODN, acting through central-type benzodiazepine receptors associated with the GABA A receptor complex, stimulates DNA synthesis in rat glial cells. These data provide evidence for an autocrine role of endozepines in the control of glial cell proliferation. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

Isomyosin expression patterns during rat heart morphogenesis: an immunohistochemical study

NARCIS (Netherlands)

de Groot, I. J.; Lamers, W. H.; Moorman, A. F.

1989-01-01

An immunohistochemical study of cardiac alpha and beta myosin heavy chain (MHC) expression during rat heart morphogenesis was performed. In tubular hearts (embryonic days, ED10-11) coexpression of both cardiac alpha and beta MHC was found throughout the heart, except for the left free wall of the

A feeder-free, human plasma-derived hydrogel for maintenance of a human embryonic stem cell phenotype in vitro

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Lewis Fiona C

2012-08-01

Full Text Available Abstract Background Human embryonic stem cells (hESCs represent a tremendous resource for cell therapies and the study of human development; however to maintain their undifferentiated state in vitro they routinely require the use of mouse embryonic fibroblast (MEF feeder-layers and exogenous protein media supplementation. Results These well established requirements can be overcome and in this study, it will be demonstrated that phenotypic stability of hESCs can be maintained using a novel, human plasma protein-based hydrogel as an extracellular culture matrix without the use of feeder cell co-culture. hESCs were resuspended in human platelet poor plasma (PPP, which was gelled by the addition of calcium containing DMEM-based hESC culture medium. Phenotypic and genomic expression of the pluripotency markers OCT4, NANOG and SOX2 were measured using immunohistochemistry and qRT-PCR respectively. Typical hESC morphology was demonstrated throughout in vitro culture and both viability and phenotypic stability were maintained throughout extended culture, up to 25 passages. Conclusions PPP-derived hydrogel has demonstrated to be an efficacious alternative to MEF co-culture with its hydrophilicity allowing for this substrate to be delivered via minimally invasive procedures in a liquid phase with polymerization ensuing in situ. Together this provides a novel technique for the study of this unique group of stem cells in either 2D or 3D both in vitro and in vivo.

Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

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Ahmed Kamel El-Sayed

2014-11-01

Full Text Available Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1 and Rex-1 (ZFP-42, zinc finger protein 42. Using embryonic stem cells (ESCs conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations.

EDA-containing fibronectin increases proliferation of embryonic stem cells.

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Noelia Losino

Full Text Available 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.

ATP-dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice.

Science.gov (United States)

Gomes, P; Chevalier, J; Boesmans, W; Roosen, L; van den Abbeel, V; Neunlist, M; Tack, J; Vanden Berghe, P

2009-08-01

The importance of dynamic interactions between glia and neurons is increasingly recognized, both in the central and enteric nervous system. However, apart from their protective role, little is known about enteric neuro-glia interaction. The aim was to investigate neuro-glia intercellular communication in a mouse culture model using optical techniques. Complete embryonic (E13) guts were enzymatically dissociated, seeded on coverslips and studied with immunohistochemistry and Ca(2+)-imaging. Putative progenitor-like cells (expressing both PGP9.5 and S-100) differentiated over approximately 5 days into glia or neurons expressing typical cell-specific markers. The glia-neuron ratio could be manipulated by specific supplements (N2, G5). Neurons and glia were functionally identified both by their Ca(2+)-response to either depolarization (high K(+)) or lysophosphatidic acid and by the expression of typical markers. Neurons responded to ACh, DMPP, 5-HT, ATP and electrical stimulation, while glia responded to ATP and ADPbetas. Inhibition of glial responses by MRS2179 suggests involvement of P2Y1 receptors. Neuronal stimulation also caused delayed glial responses, which were reduced by suramin and by exogenous apyrases that catalyse nucleotide breakdown. Conversely, glial responses were enhanced by ARL-67156, an ecto-ATPase inhibitor. In this mouse enteric co-culture, functional glia and neurons can be easily monitored using optical techniques. Glial cells can be activated directly by ATP or ADPbetas. Activation of neuronal cells (DMPP, K(+)) causes secondary responses in glial cells, which can be modulated by tuning ATP and ADP breakdown. This strongly supports the involvement of paracrine purinergic communication between enteric neurons and glia.

Nodal signals mediate interactions between the extra-embryonic and embryonic tissues in zebrafish

OpenAIRE

Xiang, Fan; Hagos, Engda G.; Xu, Bo; Sias, Christina; Kawakami, Koichi; Burdine, Rebecca D.; Dougan, Scott T.

2007-01-01

In many vertebrates, extra-embryonic tissues are important signaling centers that induce and pattern the germ layers. In teleosts, the mechanism by which the extra-embryonic yolk syncytial layer (YSL) patterns the embryo is not understood. Although the Nodal-related protein Squint is expressed in the YSL, its role in this tissue is not known. We generated a series of stable transgenic lines with GFP under the control of squint genomic sequences. In all species, nodal-related genes induce thei...

Enhanced proliferation and dopaminergic differentiation of ventral mesencephalic precursor cells by synergistic effect of FGF2 and reduced oxygen tension

DEFF Research Database (Denmark)

Jensen, Pia; Gramsbergen, Jan-Bert; Zimmer, Jens

2011-01-01

Effective numerical expansion of dopaminergic precursors might overcome the limited availability of transplantable cells in replacement strategies for Parkinson's disease. Here we investigated the effect of fibroblast growth factor-2 (FGF2) and FGF8 on expansion and dopaminergic differentiation o...... enzyme activity, which may explain the elevated dopamine levels. Our findings demonstrate that modulation of oxygen tension is a recognizable factor for in vitro expansion and dopaminergic differentiation of rat embryonic midbrain precursor cells....... of rat embryonic ventral mesencephalic neuroblasts cultured at high (20%) and low (3%) oxygen tension. More cells incorporated bromodeoxyuridine in cultures expanded at low as compared to high oxygen tension, and after 6 days of differentiation there were significantly more neuronal cells in low than......, switching FGF2-expanded cultures from low to high oxygen tension during the last two days of differentiation significantly enhanced dopamine release and intracellular dopamine levels as compared to all other treatment groups. In addition, the short-term exposure to high oxygen enhanced in situ assessed TH...

Human Embryonic Stem Cell Responses to Ionizing Radiation Exposures: Current State of Knowledge and Future Challenges

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Mykyta V. Sokolov

2012-01-01

Full Text Available Human embryonic stem cells, which are derived from the inner cell mass of the blastocyst, have become an object of intense study over the last decade. They possess two unique properties that distinguish them from many other cell types: (i the ability to self-renew indefinitely in culture under permissive conditions, and (ii the pluripotency, defined as the capability of giving rise to all cell types of embryonic lineage under the guidance of the appropriate developmental cues. The focus of many recent efforts has been on the elucidating the signaling pathways and molecular networks operating in human embryonic stem cells. These cells hold great promise in cell-based regenerative therapies, disease modeling, drug screening and testing, assessing genotoxic and mutagenic risks associated with exposures to a variety of environmental factors, and so forth. Ionizing radiation is ubiquitous in nature, and it is widely used in diagnostic and therapeutic procedures in medicine. In this paper, our goal is to summarize the recent progress in understanding how human embryonic stem cells respond to ionizing radiation exposures, using novel methodologies based on “omics” approaches, and to provide a critical discussion of what remains unknown; thus proposing a roadmap for the future research in this area.

Retinal vascular injuries and intravitreal human embryonic stem cell-derived haemangioblasts.

Science.gov (United States)

Wang, Jin-Da; An, Ying; Zhang, Jing-Shang; Wan, Xiu-Hua; Zhang, Wei; Lanza, Robert; Lu, Shi-Jiang; Jonas, Jost B; Xu, Liang

2017-09-01

To investigate whether intravitreally applied haemangioblasts (HB) derived from human embryonic stem cells (hESCs) are helpful for the repair of vascular damage caused in animals by an oxygen-induced retinopathy (OIR), by an induced diabetic retinopathy (DR) or by an induced retinal ischaemia with subsequent reperfusion. Human embryonic stem cell-derived HBs were transplanted intravitreally into C57BL/6J mice (OIR model), into male Wistar rats with an induced DR and into male Wistar rats undergoing induced retinal ischaemia with subsequent reperfusion. Control groups of animals received an intravitreal injection of endothelial cells (ECs) or phosphate-buffered saline (PBS). We examined the vasculature integrity in the mice with OIR, the blood-retina barrier in the rats with induced DR, and retinal thickness and retinal ganglion cell density in retina flat mounts of the rats with the retinal ischaemic-reperfusion retinopathy. In the OIR model, the study group versus control groups showed a significantly (p < 0.001) smaller retinal avascular area [5.1 ± 2.7%;n = 18 animals versus 12.2 ± 2.8% (PBS group; n = 10 animals) and versus 11.8 ± 3.7% (EC group; n = 8 animals)] and less retinal neovascularization [6.3 ± 2.5%;n = 18 versus 15.2 ± 6.3% (n = 10; PBS group) and versus 15.8 ± 3.3% (n = 8; EC group)]. On retinal flat mounts, hESC-HBs were integrated into damaged retinal vessels and stained positive for PECAM (CD31) as EC marker. In the DR model, the study group versus the EC control group showed a significantly (p = 0.001) better blood-retina barrier function as measured at 2 days after the intravitreal injections [study group: 20.2 ± 12.8 μl/(g × hr); n = 6; versus EC control group: 52.9 ± 9.9 μl/(g × hr; n = 6)]. In the retinal ischaemia-reperfusion model, the groups did not differ significantly in retinal thickness and retinal ganglion cell density at 2, 5 and 7 days after baseline. By integrating into

Changes in responsiveness of rat tracheal epithelial cells to growth factors during preneoplastic transformation in cell culture

International Nuclear Information System (INIS)

Thomassen, D.G.

1988-01-01

Preneoplastic rat tracheal epithelial (RTE) cell lines require fewer growth factors for clonal proliferation in culture than normal cells. Serum-free media missing various combinations of growth factors (e.g., cholera toxin, serum albumin, epidermal growth factor, hydrocortisone) required for proliferation of normal, but not preneoplastic, RTE cells can be used to select for carcinogen-induced preneoplastic variants having an increased proliferative potential in culture. These results suggest that reductions in growth factor requirements are primary events in the carcinogenic process. (author)

General developmental health in the VPA-rat model of autism

DEFF Research Database (Denmark)

Favre, Mônica R; Rinaldi Barkat, Tania; Lamendola, Deborah

2013-01-01

Autism is a neurodevelopmental condition diagnosed by impaired social interaction, abnormal communication and, stereotyped behaviors. While post-mortem and imaging studies have provided good insights into the neurobiological symptomology of autism, animal models can be used to study...... with VPA during early pregnancy show an increased risk for giving birth to an autistic child. In rats, early embryonic exposure, around the time of neural tube closure, leads to autism-like anatomical and behavioral abnormalities in the offspring. Considering the increasing use of the VPA rat model, we...

Allopregnanolone-induced rise in intracellular calcium in embryonic hippocampal neurons parallels their proliferative potential

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Brinton Roberta

2008-12-01

Full Text Available Abstract Background Factors that regulate intracellular calcium concentration are known to play a critical role in brain function and neural development, including neural plasticity and neurogenesis. We previously demonstrated that the neurosteroid allopregnanolone (APα; 5α-pregnan-3α-ol-20-one promotes neural progenitor proliferation in vitro in cultures of rodent hippocampal and human cortical neural progenitors, and in vivo in triple transgenic Alzheimer's disease mice dentate gyrus. We also found that APα-induced proliferation of neural progenitors is abolished by a calcium channel blocker, nifedipine, indicating a calcium dependent mechanism for the proliferation. Methods In the present study, we investigated the effect of APα on the regulation of intracellular calcium concentration in E18 rat hippocampal neurons using ratiometric Fura2-AM imaging. Results Results indicate that APα rapidly increased intracellular calcium concentration in a dose-dependent and developmentally regulated manner, with an EC50 of 110 ± 15 nM and a maximal response occurring at three days in vitro. The stereoisomers 3β-hydroxy-5α-hydroxy-pregnan-20-one, and 3β-hydroxy-5β-hydroxy-pregnan-20-one, as well as progesterone, were without significant effect. APα-induced intracellular calcium concentration increase was not observed in calcium depleted medium and was blocked in the presence of the broad spectrum calcium channel blocker La3+, or the L-type calcium channel blocker nifedipine. Furthermore, the GABAA receptor blockers bicuculline and picrotoxin abolished APα-induced intracellular calcium concentration rise. Conclusion Collectively, these data indicate that APα promotes a rapid, dose-dependent, stereo-specific, and developmentally regulated increase of intracellular calcium concentration in rat embryonic hippocampal neurons via a mechanism that requires both the GABAA receptor and L-type calcium channel. These data suggest that AP�

Altered glucose transport to utero-embryonic unit in relation to delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx.

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Arnab, Banerjee; Amitabh, Krishna

2011-02-10

The aim of this study was to compare the changes in concentration of glucose and glucose transporters (GLUTs) in the utero-embryonic unit, consisting of decidua, trophoblast and embryo, during delayed and non-delayed periods to understand the possible cause of delayed embryonic development in Cynopterus sphinx. The results showed a significantly decreased concentration of glucose in the utero-embryonic unit due to decline in the expression of insulin receptor (IR) and GLUT 3, 4 and 8 proteins in the utero-embryonic unit during delayed period. The in vitro study showed suppressive effect of insulin on expression of GLUTs 4 and 8 in the utero-embryonic unit and a significant positive correlation between the decreased amount of glucose consumed by the utero-embryonic unit and decreased expression of GLUTs 4 (r=0.99; psphinx. Increased supply of fatty acid to the delayed embryo may be responsible for its survival under low glucose condition but unable to promote embryonic development in C. sphinx. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Transcriptomics-based identification of developmental toxicants through their interference with cardiomyocyte differentiation of embryonic stem cells

International Nuclear Information System (INIS)

Dartel, Dorien A.M. van; Pennings, Jeroen L.A.; Schooten, Frederik J. van; Piersma, Aldert H.

2010-01-01

The embryonic stem cell test (EST) predicts developmental toxicity based on the inhibition of cardiomyocyte differentiation of embryonic stem cells (ESC). The subjective endpoint, the long culture duration together with the undefined applicability domain and related predictivity need further improvement to facilitate implementation of the EST into regulatory strategies. These aspects may be improved by studying gene expression changes in the ESC differentiation cultures and their modulation by compound exposure using transcriptomics. Here, we tested the developmental toxicants monobutyl phthalate and 6-aminonicotinamide. ESC were allowed to differentiated, and cardiomyocyte differentiation was assessed after 10 days of culture. RNA of solvent controls was collected after 0, 24, 48, 72 and 96 h of exposure, and RNA of developmental-toxicant-exposed cultures was collected after 24 and 96 h. Samples were hybridized to DNA microarrays, and 1355 genes were found differentially expressed among the unexposed experimental groups. These regulated genes were involved in differentiation-related processes, and Principal Component Analysis (PCA) based on these genes showed that the unexposed experimental groups appeared in chronological order in the PCA, which can therefore be regarded as a continuous representation of the differentiation track. The developmental-toxicant-exposed cultures appeared to deviate significantly from this differentiation track, which confirms the compound-modulating effects on the differentiation process. The incorporation of transcriptomics in the EST is expected to provide a more informative and improved endpoint in the EST as compared with morphology, allowing early detection of differentiation modulation. Furthermore, this approach may improve the definition of the applicability domain and predictivity of the EST.

CHANGES IN THE GLUTATHIONE SYSTEM IN P19 EMBRYONAL CARCINOMA CELLS UNDER HYPOXIC CONDITIONS

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D. S. Orlov

2015-01-01

Full Text Available Introduction. According to modern perceptions, tumor growth, along with oxidative stress formation, is accompanied by hypoxia. Nowadays studying the regulation of cellular molecular system functioning by conformational changes in proteins appears to be a topical issue. Research goal was to evaluate the state of the glutathione system and the level of protein glutathionylation in P19 embryonal carcinoma (EC cells under hypoxic conditions.Material and methods. P19 EC cells (mouse embryonal carcinoma cultured under normoxic and hypox-ic conditions served the research material.The concentration of total, oxidized, reduced and protein-bound glutathione, the reduced to oxidized thiol ratio as well as glutathione peroxidase and glutathione reductase activity were determined by spectropho-tometry.Results. Glutathione imbalance was accompanied by a decrease in P19 EC cell redox status under hypox-ic conditions against the backdrop of a rise in protein-bound glutathione.Conclusions. As a result of the conducted study oxidative stress formation was identified when modeling hypoxia in P19 embryonal carcinoma cells. The rise in the concentration of protein-bound glutathione may indicate the role of protein glutathionylation in regulation of P19 cell metabolism and functions un-der hypoxia. 

Gelatin–PMVE/MA composite scaffold promotes expansion of embryonic stem cells

International Nuclear Information System (INIS)

Chhabra, Hemlata; Gupta, Priyanka; Verma, Paul J.; Jadhav, Sameer; Bellare, Jayesh R.

2014-01-01

We introduce a new composite scaffold of gelatin and polymethyl vinyl ether-alt-maleic anhydride (PMVE/MA) for expansion of embryonic stem cells (ESCs) in an in vitro environment. To optimize the scaffold, we prepared a gelatin scaffold (G) and three composite scaffolds namely GP-1, GP-2, and GP-3 with varying PMVE/MA concentrations (0.2–1%) and characterized them by scanning electron microscopy (SEM), swelling study, compression testing and FTIR. SEM micrographs revealed interconnected porous structure in all the scaffolds. The permissible hemolysis ratio and activation of platelets by scaffolds confirmed the hemocompatibility of scaffolds. Initial biocompatibility assessment of scaffolds was conducted using hepatocarcinoma (Hep G2) cells and adhesion, proliferation and infiltration of Hep G2 cells in depth of scaffolds were observed, proving the scaffold's biocompatibility. Further Oct4B2 mouse embryonic stem cells (mESCs), which harbor a green fluorescence protein transgene under regulatory control of the Oct4 promotor, were examined for expansion on scaffolds with MTT assay. The GP-2 scaffold demonstrated the best cell proliferation and was further explored for ESC adherence and infiltration in depth (SEM and confocal), and pluripotent state of mESCs was assessed with the expression of Oct4-GFP and stage-specific embryonic antigen-1 (SSEA-1). This study reports the first demonstration of biocompatibility of gelatin–PMVE/MA composite scaffold and presents this scaffold as a promising candidate for embryonic stem cell based tissue engineering. - Highlights: • Composite scaffolds of gelatin and PMVE/MA were prepared by freeze-drying method. • SEM micrographs showed porous structure in all scaffolds of varying pore dimension. • GP-2 composite exhibited better cellular response in comparison to other scaffolds. • mESCs proliferated and expressed Oct-4 and SSEA-1, when cultured on GP-2 scaffold

Gelatin–PMVE/MA composite scaffold promotes expansion of embryonic stem cells

Energy Technology Data Exchange (ETDEWEB)

Chhabra, Hemlata [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); Gupta, Priyanka [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); IITB-Monash Research Academy, Mumbai (India); Department of Chemical Engineering, Monash University, Melbourne (Australia); Verma, Paul J. [Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, South Australia (Australia); Jadhav, Sameer; Bellare, Jayesh R. [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India)

2014-04-01

We introduce a new composite scaffold of gelatin and polymethyl vinyl ether-alt-maleic anhydride (PMVE/MA) for expansion of embryonic stem cells (ESCs) in an in vitro environment. To optimize the scaffold, we prepared a gelatin scaffold (G) and three composite scaffolds namely GP-1, GP-2, and GP-3 with varying PMVE/MA concentrations (0.2–1%) and characterized them by scanning electron microscopy (SEM), swelling study, compression testing and FTIR. SEM micrographs revealed interconnected porous structure in all the scaffolds. The permissible hemolysis ratio and activation of platelets by scaffolds confirmed the hemocompatibility of scaffolds. Initial biocompatibility assessment of scaffolds was conducted using hepatocarcinoma (Hep G2) cells and adhesion, proliferation and infiltration of Hep G2 cells in depth of scaffolds were observed, proving the scaffold's biocompatibility. Further Oct4B2 mouse embryonic stem cells (mESCs), which harbor a green fluorescence protein transgene under regulatory control of the Oct4 promotor, were examined for expansion on scaffolds with MTT assay. The GP-2 scaffold demonstrated the best cell proliferation and was further explored for ESC adherence and infiltration in depth (SEM and confocal), and pluripotent state of mESCs was assessed with the expression of Oct4-GFP and stage-specific embryonic antigen-1 (SSEA-1). This study reports the first demonstration of biocompatibility of gelatin–PMVE/MA composite scaffold and presents this scaffold as a promising candidate for embryonic stem cell based tissue engineering. - Highlights: • Composite scaffolds of gelatin and PMVE/MA were prepared by freeze-drying method. • SEM micrographs showed porous structure in all scaffolds of varying pore dimension. • GP-2 composite exhibited better cellular response in comparison to other scaffolds. • mESCs proliferated and expressed Oct-4 and SSEA-1, when cultured on GP-2 scaffold.

YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers

DEFF Research Database (Denmark)

Brøchner, Christian B; Johansen, Julia S; Larsen, Lars A

2012-01-01

oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted......The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high...... YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3ß, PDX1, CD34, p63, nestin, PAX6) markers. Double...

Elastase effect on the extracellular matrix of rat aortic smooth muscle cells in culture

International Nuclear Information System (INIS)

Kispert, J.; Mogayzel, P.J. Jr.; Pratt, C.A.; Toselli, P.; Wolfe, B.L.; Faris, B.; Franzblau, C.

1986-01-01

The effect of porcine pancreatic elastase on the extracellular matrix (ECM) of neonatal rat aortic smooth muscle cell cultures was monitored both chemically and ultrastructurally. Initially, the elastin appeared as non-coalesced material closely associated with filaments, presumably microfibrils. The insoluble elastin accumulated in the ECM of cells in culture for 6 weeks accounted for 40-45% of the total protein. After exposure to elastase for 30-60 minutes, the elastin content was reduced to 14-20%. The reduction in the total protein content of the cultures after elastase treatment was due primarily to the loss of elastin. Although the amino acid compositions of the elastin isolated from cultures both before and after elastase treatment were similar, there were striking ultrastructural differences in the amorphous elastin. The elastin assumed a mottled appearance after elastase exposure, similar to that seen in in vivo emphysema models. Pulse experiments with 3 H-valine demonstrated an increase in protein synthesis by the cells 20 hours after elastase exposure, suggesting the potential for elastin repair. The use of this culture system will aid in clarifying the role of elastolysis in pulmonary and vascular injuries

Characterization of cell cultures derived from Lutzomyia spinicrassa (Diptera: Psychodidae) and their susceptibility to infection with Leishmania (Viannia) braziliensis.

Science.gov (United States)

Zapata Lesmes, Angela Cristina; Cárdenas Castro, Estrella; Bello, Felio

2005-12-01

The sand fly Lutzomyia spinicrassa (Morales, Osorno-Mesa, Osorno & de Hoyos, 1969) is a vector of Leishmania (Viannia) braziliensis, an etiological agent of cutaneous leishmaniasis in Colombia. The present article describes, for the first time, the morphological, karyotypical, and isozymatic characteristics of cell cultures derived from L. Spinicrassa embryonic tissues as well as the interaction of L. Braziliensis with these cell cultures. L. Spinicrassa embryonated eggs and neonate larvae were taken for tissue explants. These were seeded in Grace, L-15, Grace/L-15, MM/VP12, and MK/VP12 culture media. The pH range in these media was 6.7 to 6.9 and the cultures were incubated at 28 degrees C. The MHOM/CO/86/CL250 strain of L. Braziliensis was used for experimental infection of cell cultures of L. Spinicrassa. Cell growth was achieved in L-15 medium and a confluent monolayer was obtained 180 days after the embryonated eggs were explanted. The cell morphology of the primary cell cultures was initially heterogeneous, but in the confluent monolayer of these cell cultures and in the subcultures the predominant cell types were later fibroblast-like and epithelial-like. Cultured cells were predominantly diploid (2n=8); however, significant percentages of aneuploids were also recorded. The cell culture isozyme patterns of L. Spinicrassa coincided with pupae samples from the same species. Promastigote forms of L. Braziliensis could invade cells and transform into amastigote-like forms inside them. The characteristics of cell cultures derived from L. Spinicrassa embryonic tissues were determined. These cultures emerge as a new model to study the life-cycle of L. Braziliensis.

Characterization of bovine embryos cultured under conditions appropriate for sustaining human naïve pluripotency

NARCIS (Netherlands)

Brinkhof, Bas; van Tol, Helena T A; Groot Koerkamp, Marian J A; Wubbolts, Richard W; Haagsman, Henk P; Roelen, Bernard A J

2017-01-01

In mammalian preimplantation development, pluripotent cells are set aside from cells that contribute to extra-embryonic tissues. Although the pluripotent cell population of mouse and human embryos can be cultured as embryonic stem cells, little is known about the pathways involved in formation of a

Suppression of sterol 27-hydroxylase mRNA and transcriptional activity by bile acids in cultured rat hepatocytes

NARCIS (Netherlands)

Twisk, J.; Wit, E.C.M. de; Princen, H.M.G.

1995-01-01

In previous work we have demonstrated suppression of cholesterol 7α-hydroxylase by bile acids at the level of mRNA and transcription, resulting in a similar decline in bile acid synthesis in cultured rat hepatocytes. In view of the substantial contribution of the 'alternative' or '27-hydroxylase'

The 'ventral organs' of Pycnogonida (Arthropoda) are neurogenic niches of late embryonic and post-embryonic nervous system development.

Science.gov (United States)

Brenneis, Georg; Scholtz, Gerhard

2014-01-01

Early neurogenesis in arthropods has been in the focus of numerous studies, its cellular basis, spatio-temporal dynamics and underlying genetic network being by now comparably well characterized for representatives of chelicerates, myriapods, hexapods and crustaceans. By contrast, neurogenesis during late embryonic and/or post-embryonic development has received less attention, especially in myriapods and chelicerates. Here, we apply (i) immunolabeling, (ii) histology and (iii) scanning electron microscopy to study post-embryonic ventral nerve cord development in Pseudopallene sp., a representative of the sea spiders (Pycnogonida), the presumable sister group of the remaining chelicerates. During early post-embryonic development, large neural stem cells give rise to additional ganglion cell material in segmentally paired invaginations in the ventral ectoderm. These ectodermal cell regions - traditionally designated as 'ventral organs' - detach from the surface into the interior and persist as apical cell clusters on the ventral ganglion side. Each cluster is a post-embryonic neurogenic niche that features a tiny central cavity and initially still houses larger neural stem cells. The cluster stays connected to the underlying ganglionic somata cortex via an anterior and a posterior cell stream. Cell proliferation remains restricted to the cluster and streams, and migration of newly produced cells along the streams seems to account for increasing ganglion cell numbers in the cortex. The pycnogonid cluster-stream-systems show striking similarities to the life-long neurogenic system of decapod crustaceans, and due to their close vicinity to glomerulus-like neuropils, we consider their possible involvement in post-embryonic (perhaps even adult) replenishment of olfactory neurons - as in decapods. An instance of a potentially similar post-embryonic/adult neurogenic system in the arthropod outgroup Onychophora is discussed. Additionally, we document two transient posterior

Oral Morphine Consumption Reduces Lens Development in Rat Embryos

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Hossein Bahadoran

2012-07-01

Full Text Available Objective: Consumption of morphine, during pregnancy, in addition to inducing defects in the mother’s nervous system function, caused defects or delays in the formation and evolution of embryonic visual system. In the present study, changes in lens development was assessed in embryos exposed in utero to morphine. Material and Methods: Female Wistar rats (250-300 g were mated with male rats and pregnancy was determined by sperm observation in vaginal smear. This day was considered as embryonic day zero (E0. The females were then divided randomly into the experimental and the control groups. The control group received tap water and the experimental group received morphine (0.05 mg/ml in their water. On embryonic day 13 ( E13, blood samples were collected from the retro-orbital sinus of all animals for plasma corticosterone detection. On embryonic day 17(E17, the animals were killed by an overdose of chloroform and the embryos were taken out surgically. The embryos were fixed in 10% formalin for 30 days. At this time, the head of the embryos were removed for tissue processing and Hematoxylin- Eosin (H&E staining. The samples were evaluated using light microscope and MOTIC software. Results: Our data indicated that plasma corticosterone level was dramatically increased and the lens was thinner in the experimental group. (Although the proliferation of lens cells increased in the experiment group but that lens had delay in removing the proliferated and elongation cells with abnormal density in the lateral part of the lens in compare with control group. I have no idea what the authors are stating here. Moreover, the opening of the eyelids was delayed in the off springs of the mothers who received morphine. Conclusions: This study showed that morphine consumption during pregnancy leads to defects in fetal visual system development, particularly in the lens, and eyelids.

Cytokines effects on radio-induced apoptosis in cortical and hippocampal rat cells in culture

International Nuclear Information System (INIS)

Coffigny, H.; Briot, D.; Le Nin, I.

2000-01-01

In the central nervous system in development the radio-induced cell death occurs mainly by apoptosis. The effects of modulating factors like cytokines were studied on this kind of death. To handle more easily parameters implicated in nerve cell apoptosis, we studied the effects of radiation with a in vitro system. Cells were isolated from rat foetal cortex and hippocampus, two of the major structures implicated in human mental retardation observed after exposition in utero at Hiroshima and Nagasaki. Cortical or hippocampal cells were isolated from 17 day-old rat foetuses by enzymatic and mechanical treatments and irradiated with 0.50 or 1 Gy. The cells from both structures were cultured 1 or 3 days in serum free medium. Cytokines like βNGF, NT3, EGF, βTGF, α and βFGF, IGF I and II, interleukines like Il 1β, Il 2 and IL 6 were added to the medium. In 3 days cortical cell culture, only βFGF increased cell survival with as little as 10 ng/ml. This effect was dose dependent. In hippocampal cell culture, no significant increase of cell survival occurred with 10 ng/ml of any cytokines. In the same system culture with 1 Gy irradiation, the positive or negative effect of the association of βFGF with another cytokine was tested on cell survival. Only the association with EGF induced higher cell survival in cortical cell culture. In hippocampal cell culture where βFGF alone had no effect, the cell survival was not modified by the association. In the same system, the triple association of βFGF-EGF with another cytokine was tested on hippocampal and cortical cell cultures. No significant effect was observed in both cultures but cell survival trented to decrease with βTGF. In order to avoid the mitotic effect of cytokines in the 3 day-old culture, experiments were carried out on 20 hours cell culture, before the end of the first round of the cell cycle, with the selected cytokines (βFGF or βFGF-EGF). Without irradiation, the percentage of cortical cell survival

The influence of temperature on the embryonic development of the annual fish Cynopoecilus melanotaenia (Cyprinodontiformes, Rivulidae

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A. ARENZON

Full Text Available The present study aims to provide data about the time required for Cynopoecilus melanotaenia kept at different temperatures to complete embryonic development. This information can be valuable for optimizing laboratory culture and facilitating future use of this species as a test organism in toxicity tests. Temperature effects on hatching rate are presented as well as information related to embryonic development stages. Eggs were observed daily, from start to finish of embryonic development. Thirteen developmental stages were described. Eggs were kept at two constant temperatures (20°C and 25°C and at a variable ambient temperature (16-25°C - mean = 21°C, sd = 1.95, to determine developmental rate (velocity at each temperature. A shorter incubation period was necessary to complete development at 25° ± 1°C. However, all embryos kept at this temperature hatched with morphological defects, which prevented their survival. No significant difference in developmental time period (p = 0.05 was observed at the 20°C and 16°-25°C (mean = 21°C, sd = 1.95 temperatures.

U.V.-enhanced reactivation of u.v.-irradiated herpes virus by primary cultures of rat hepatocytes

International Nuclear Information System (INIS)

Zurlo, J.; Yager, J.D.

1984-01-01

Carcinogen treatment of cultured mammalian cells prior to infection with u.v.-irradiated virus results in enhanced virus survival and mutagenesis suggesting the induction of SOS-type processes. In this paper, we report the development of a primary rat hepatocyte culture system to investigate cellular responses to DNA damage which may be relevant to hepatocarcinogenesis in vivo. We have obtained data demonstrating that enhanced reactivation of u.v.-irradiated Herpes simplex virus type 1 (HSV-1) occurs in hepatocytes irradiated with u.v. Cultured hepatocytes were pretreated with u.v. at the time of enhanced DNA synthesis. These treatments caused an inhibition followed by a recovery of DNA synthesis. At various times after pretreatment, the hepatocytes were infected with control or u.v.-irradiated HSV-1 at low multiplicity, and virus survival was measured by direct plaque assay. U.v.-irradiated HSV-1 exhibited the expected two-component survival curve in control or u.v. pretreated hepatocytes. The magnitude of enhanced reactivation of HSV-1 was dependent on the u.v. dose to the hepatocytes, the time of infection following u.v. pretreatment, and the level of DNA synthesis at the time of pretreatment. These results suggest that u.v. treatment of rat hepatocytes causes the induction of SOS-type functions that may have a role in the initiation of hepatocarcinogenesis

Neural stem cell isolation and culture from C57BL/6 mice

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S Koirala

2015-07-01

Full Text Available INTRODUCTION A widely used in vitro culture, the neurosphere assay (NSA has provided a means to retrospectively identify neural progenitor cells as well as to determine both their selfrenewal capacity. Objective of study was to isolate and compare growth of the embryonic neuronal stem cell and adult neuronal stem cells in presence of Epidermal Growth Factor (EGF and Fibroblastic Growth Factor (FGF2. MATERIALS AND METHODS Embryonic neuronal stem cell were collected from cortical plate of dorsal telencephalon of fifteen C57BL/6 transgenic mice using stereoscopic microscope on 11th gestational day (GD. Adult mammalian neuronal stem cells taken from subventricular zone (SVZ of the lateral ventricles and subgranular layer of the dentate gyrus of the hippocampus were cultured. The growth for the neurosphere was then observed in interval of 24 and 72 hours. RESULT The adult stem cell culture showed few intact cells with high amount of debris and 9% heterogeneous sphere after 24 hours while only 20 % was observed at the end of 72 hours. Higher proliferation rate was observed in embryonic neurospheres than the adult stem cell culture. CONCLUSION Presence of EGF and basic FGF2 is essential for culture of neurospheres.DOI: http://dx.doi.org/10.3126/jcmsn.v10i2.12946 Journal of College of Medical Sciences-Nepal, 2014, Vol.10(2; 1-3

Differentiation of monkey embryonic stem cells to hepatocytes by feeder-free dispersion culture and expression analyses of cytochrome p450 enzymes responsible for drug metabolism.

Science.gov (United States)

Maruyama, Junya; Matsunaga, Tamihide; Yamaori, Satoshi; Sakamoto, Sakae; Kamada, Noboru; Nakamura, Katsunori; Kikuchi, Shinji; Ohmori, Shigeru

2013-01-01

We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.

Staurosporine induces different cell death forms in cultured rat astrocytes

International Nuclear Information System (INIS)

Simenc, Janez; Lipnik-Stangelj, Metoda

2012-01-01

Astroglial cells are frequently involved in malignant transformation. Besides apoptosis, necroptosis, a different form of regulated cell death, seems to be related with glioblastoma genesis, proliferation, angiogenesis and invasion. In the present work we elucidated mechanisms of necroptosis in cultured astrocytes, and compared them with apoptosis, caused by staurosporine. Cultured rat cortical astrocytes were used for a cell death studies. Cell death was induced by different concentrations of staurosporine, and modified by inhibitors of apoptosis (z-vad-fmk) and necroptosis (nec-1). Different forms of a cell death were detected using flow cytometry. We showed that staurosporine, depending on concentration, induces both, apoptosis as well as necroptosis. Treatment with 10 −7 M staurosporine increased apoptosis of astrocytes after the regeneration in a staurosporine free medium. When caspases were inhibited, apoptosis was attenuated, while necroptosis was slightly increased. Treatment with 10 −6 M staurosporine induced necroptosis that occurred after the regeneration of astrocytes in a staurosporine free medium, as well as without regeneration period. Necroptosis was significantly attenuated by nec-1 which inhibits RIP1 kinase. On the other hand, the inhibition of caspases had no effect on necroptosis. Furthermore, staurosporine activated RIP1 kinase increased the production of reactive oxygen species, while an antioxidant BHA significantly attenuated necroptosis. Staurosporine can induce apoptosis and/or necroptosis in cultured astrocytes via different signalling pathways. Distinction between different forms of cell death is crucial in the studies of therapy-induced necroptosis

The in vitro biokinetics of chlorpromazine and diazepam in aggregating rat brain cell cultures after repeated exposure

NARCIS (Netherlands)

Broeders, Jessica J W; Hermens, Joop L M; Blaauboer, Bas J; Zurich, Marie-Gabrielle

2015-01-01

Neurotoxic effects of compounds can be tested in vitro using cell systems. One example is aggregating rat brain cell cultures. For the extrapolation of in vitro data to the in vivo situation, it is important to take the biokinetics of the test compound into account. In addition, the exposure in vivo

Hypophyseal corticosteroids stimulate somatotrope differentiation in the embryonic chicken pituitary gland.

Science.gov (United States)

Zheng, Jun; Takagi, Hiroyasu; Tsutsui, Chihiro; Adachi, Akihito; Sakai, Takafumi

2008-03-01

Although it is known that glucocorticoids induce differentiation of growth hormone (GH)-producing cells in rodents and birds, the effect of mineralocorticoids on GH mRNA expression and the origin of corticosteroids affecting somatotrope differentiation have not been elucidated. In this study, we therefore carried out experiments to determine the effect of mineralocorticoids on GH mRNA expression in the chicken anterior pituitary gland in vitro and to determine whether corticosteroids are synthesized in the chicken embryonic pituitary gland. In a pituitary culture experiment with E11 embryos, both corticosterone and aldosterone stimulated GH mRNA expression and increased the number of GH cells in both lobes of the pituitary gland in a dose-dependent manner. These effects of the corticosteroids were significantly reversed by pretreatment with mifepristone, a glucocorticoid receptor (GR) antagonist, or spironolactone, a mineralocorticoid receptor (MR) antagonist. Interestingly, an in vitro serum-free culture experiment with an E11 pituitary gland showed that the GH mRNA level spontaneously increased during cultivation for 2 days without any extra stimulation, and this increase in GH mRNA level was completely suppressed by metyrapone, a corticosterone-producing enzyme P450C11 inhibitor. Moreover, progesterone, the corticosterone precursor, also stimulated GH mRNA expression in the cultured chicken pituitary gland, and this effect was blocked by pretreatment with metyrapone. We also detected mRNA expression of enzymes of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase1 (3beta-HSD1) in the developmental chicken pituitary gland from E14 and E18, respectively. These results suggest that mineralocorticoids as well as glucocorticoids can stimulate GH mRNA expression and that corticosteroids generated in the embryonic pituitary gland by intrinsic steroidogenic enzymes stimulate somatotrope differentiation.

Isolation and culture of larval cells from C. elegans.

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Sihui Zhang

Full Text Available Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×10(4 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81% of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology.

Transplantation of embryonic porcine neocortical tissue into newborn rats

DEFF Research Database (Denmark)

Castro, Anthony J; Meyer, Morten; Møller Dall, Annette

2003-01-01

cavities made in newborn rats. Sacrifice at 3-12.5 weeks posttransplantation revealed healthy looking grafts in several animals. Apparent graft rejection evidenced by areas of necrosis and OX1 reactivity was observed in some of the older transplants. Treatment of nursing mothers or of postweaning newborns...... with cyclosporin A did not appear to promote graft survival. Some transplants grew to extremely large proportions and were characterized by bands of cells and bundles of axons as observed using immunohistochemical staining for pig neurofilament. Neurofilament-positive axons projected from several of the grafts...

Effects of Feeder Cells on Dopaminergic Differentiation of Human Embryonic Stem Cells

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Zhenqiang Zhao

2016-12-01

Full Text Available Mouse embryonic fibroblasts (MEFs and human foreskin fibroblasts (HFFs are used for the culture of human embryonic stem cells (hESCs. MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation, two hESCs lines were cultured on mixed feeder cells (MFCs, MEFs: HFFs =1:1 and HFFs feeder respectively, and then were differentiated into DA neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry, quantitative fluorescent real-time PCR (qRT-PCR, transmission and scanning electron microscopy, and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However, compared to hESCs line on MFCs feeder, hESCs line on HFFs feeder had a higher proportion of TH positive cells and expressed higher levels of FOXA2, PITX3, NURR1 and TH genes. In addition, the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion, HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons, but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore, feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines, but also electrophysiological properties of hESCs-derived DA neurons.

CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

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Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

2010-01-01

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

Influence of flow conditions and matrix coatings on growth and differentiation of three-dimensionally cultured rat hepatocytes.

Science.gov (United States)

Fiegel, Henning C; Havers, Joerg; Kneser, Ulrich; Smith, Molly K; Moeller, Tim; Kluth, Dietrich; Mooney, David J; Rogiers, Xavier; Kaufmann, Peter M

2004-01-01

Maintenance of liver-specific function of hepatocytes in culture is still difficult. Improved culture conditions may enhance the cell growth and function of cultured cells. We investigated the effect of three-dimensional culture under flow conditions, and the influence of surface modifications in hepatocyte cultures. Hepatocytes were harvested from Lewis rats. Cells were cultured on three-dimensional polymeric poly-lactic-co-glycolic acid (PLGA) matrices in static culture, or in a pulsatile flow-bioreactor system. Different surface modifications of matrices were investigated: coating with collagen I, collagen IV, laminin, or fibronectin; or uncoated matrix. Hepatocyte numbers, DNA content, and albumin secretion rate were assessed over the observation period. Culture under flow condition significantly enhanced cell numbers. An additional improvement of this effect was observed, when matrix coating was used. Cellular function also showed a significant increase (4- to 5-fold) under flow conditions when compared with static culture. Our data showed that culture under flow conditions improves cell number, and strongly enhances cellular function. Matrix modification by coating with extracellular matrix showed overall an additive stimulatory effect. Our conclusion is that combining three-dimensional culture under flow conditions and using matrix modification significantly improves culture conditions and is therefore attractive for the development of successful culture systems for hepatocytes.

Effect of human vascular endothelial growth factor gene transfer on endogenous vascular endothelial growth factor mRNA expression in a rat fibroblast and osteoblast culture model.

Science.gov (United States)

Li, Ru; Li, Claire H; Nauth, Aaron; McKee, Michael D; Schemitsch, Emil H

2010-09-01

Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point

Intermedin attenuates LPS-induced inflammation in the rat testis.

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Lei Li

Full Text Available First reported as a vasoactive peptide in the cardiovascular system, intermedin (IMD, also known as adrenomedullin 2 (ADM2, is a hormone with multiple potent roles, including its antioxidant action on the pulmonary, central nervous, cardiovascular and renal systems. Though IMD may play certain roles in trophoblast cell invasion, early embryonic development and cumulus cell-oocyte interaction, the role of IMD in the male reproductive system has yet to be investigated. This paper reports our findings on the gene expression of IMD, its receptor components and its protein localization in the testes. In a rat model, bacterial lippolysaccharide (LPS induced atypical orchitis, and LPS treatment upregulated the expression of IMD and one of its receptor component proteins, i.e. receptor activity modifying protein 2 (RAMP2. IMD decreased both plasma and testicular levels of reactive oxygen species (ROS production, attenuated the increase in the gene expression of the proinflammatory cytokines tumor necrosis factor alpha (TNFα, interleukin 6 (IL6 and interleukin 1 beta (IL1β, rescued spermatogenesis, and prevented the decrease in plasma testosterone levels caused by LPS. The restorative effect of IMD on steroidogenesis was also observed in hydrogen peroxide-treated rat primary Leydig cells culture. Our results indicate IMD plays an important protective role in spermatogenesis and steroidogenesis, suggesting therapeutic potential for IMD in pathological conditions such as orchitis.

Oxygen-controlled automated neural differentiation of mouse embryonic stem cells.

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Mondragon-Teran, Paul; Tostoes, Rui; Mason, Chris; Lye, Gary J; Veraitch, Farlan S

2013-03-01

Automation and oxygen tension control are two tools that provide significant improvements to the reproducibility and efficiency of stem cell production processes. the aim of this study was to establish a novel automation platform capable of controlling oxygen tension during both the cell-culture and liquid-handling steps of neural differentiation processes. We built a bespoke automation platform, which enclosed a liquid-handling platform in a sterile, oxygen-controlled environment. An airtight connection was used to transfer cell culture plates to and from an automated oxygen-controlled incubator. Our results demonstrate that our system yielded comparable cell numbers, viabilities, metabolism profiles and differentiation efficiencies when compared with traditional manual processes. Interestingly, eliminating exposure to ambient conditions during the liquid-handling stage resulted in significant improvements in the yield of MAP2-positive neural cells, indicating that this level of control can improve differentiation processes. This article describes, for the first time, an automation platform capable of maintaining oxygen tension control during both the cell-culture and liquid-handling stages of a 2D embryonic stem cell differentiation process.

The influence of serum substituents on serum-free Vero cell conditioned culture media manufactured from Dulbecco's modified Eagle medium in mouse embryo culture.

Science.gov (United States)

Lee, Jong-Seon; Kim, Ju-Hwan; Seo, Young-Seok; Yang, Jung-Bo; Kim, Yong-Il; Kim, Hye-Jin; Lee, Ki-Hwan

2013-09-01

This study was conducted to examine the influences of supplementation of the serum substituents and available period of serum-free Vero cell conditioned media (SF-VCM) manufactured from Dulbecco's modified Eagle medium cultured with Vero cells for in vitro development of mouse preimplantation embryos. A total of 1,099 two-cell embryos collected from imprinting control region mice were cultured in SF-VCM with 10% and 20% human follicular fluid (hFF), serum substitute supplement (SSS), and serum protein substitute (SPS). Development of embryos was observed every 24 hours. Results between different groups were analyzed by chi-square test, and considered statistically significant when P-value was less than 0.05. The rates of embryonic development cultured in SF-VCM supplemented with serum substituents were significantly higher compare with serum-free group (P media up to 4 weeks did not affect on embryonic development.

In vitro expansion and differentiation of rat pancreatic duct-derived stem cells into insulin secreting cells using a dynamicthree-dimensional cell culture system.

Science.gov (United States)

Chen, X C; Liu, H; Li, H; Cheng, Y; Yang, L; Liu, Y F

2016-06-27

In this study, a dynamic three-dimensional cell culture technology was used to expand and differentiate rat pancreatic duct-derived stem cells (PDSCs) into islet-like cell clusters that can secrete insulin. PDSCs were isolated from rat pancreatic tissues by in situ collagenase digestion and density gradient centrifugation. Using a dynamic three-dimensional culture technique, the cells were expanded and differentiated into functional islet-like cell clusters, which were characterized by morphological and phenotype analyses. After maintaining 1 x 108 isolated rat PDSCs in a dynamic three-dimensional cell culture for 7 days, 1.5 x 109 cells could be harvested. Passaged PDSCs expressed markers of pancreatic endocrine progenitors, including CD29 (86.17%), CD73 (90.73%), CD90 (84.13%), CD105 (78.28%), and Pdx-1. Following 14 additional days of culture in serum-free medium with nicotinamide, keratinocyte growth factor (KGF), and b fibroblast growth factor (FGF), the cells were differentiated into islet-like cell clusters (ICCs). The ICC morphology reflected that of fused cell clusters. During the late stage of differentiation, representative clusters were non-adherent and expressed insulin indicated by dithizone (DTZ)-positive staining. Insulin was detected in the extracellular fluid and cytoplasm of ICCs after 14 days of differentiation. Additionally, insulin levels were significantly higher at this time compared with the levels exhibited by PDSCs before differentiation (P cell culture system, PDSCs can be expanded in vitro and can differentiate into functional islet-like cell clusters.

Regeneration of plantlets from unpollinated ovary cultures of ...

African Journals Online (AJOL)

Tsega

2013-09-25

Sep 25, 2013 ... 3Ethiopian Institute of Agricultural Research (EIAR): Holetta ... cultured unpollinated ovaries, 1,100 embryonic tissues (7.6 %) and 75 regenerants were obtained. The ... viral diseases, insect pests and increasing human popu-.

Effect of sucralfate and its components on taurocholate-induced damage to rat gastric mucosal cells in tissue culture

Energy Technology Data Exchange (ETDEWEB)

Romano, M.; Razandi, M.; Ivey, K.J. (Long Beach VA Medical Center, CA (USA))

1990-04-01

The present study evaluated the effect of sucralfate and its components, sucrose octasulfate and aluminum hydroxide, on: (1) damage to rat cultured gastric mucosal cells induced by sodium taurocholate in a neutral environment and in conditions independent of systemic factors, (2) prostaglandin E2 and on 6-keto prostaglandin F1 alpha release by cultured cells, and (3) sulfhydryl content of cultured cells. Cell damage was quantitated by chromium-51 release assay. Prostaglandin E2 and 6-keto prostaglandin F1 alpha were measured by radioimmunoassay. Total sulfhydryl content of cultured cells was determined calorimetrically. Microscopically, sucralfate was found to adhere tightly to epithelial cell surfaces despite frequent washings. Sucralfate 2 mg/ml and 5 mg/ml significantly decreased taurocholate-induced damage, reducing taurocholate-induced specific 51Cr release by 11.8 points (equal to 29% decrease in cell damage, P less than 0.01) and 22.9 points (equal to 56% decrease in cell damage, P less than 0.001), respectively. Sucrose octasulfate and aluminum hydroxide did not exert significant protection against damage induced by sodium taurocholate. The protective effect of sucralfate was not prevented by indomethacin, nor was it counteracted by the sulfhydryl blocker, iodoacetamide. Sucralfate, but not its components, significantly and dose-dependently stimulated prostaglandin E2 (r = 0.94, P less than 0.05) and 6-keto prostaglandin F1 alpha (r = 0.89, P less than 0.05) production by cultured cells. Neither sucralfate nor its components affected sulfhydryl content of cultured cells. In conclusion, sucralfate, but not its components, (1) protects rat gastric mucosal cells against taurocholate-induced damage in conditions independent of systemic factors and in a neutral environment and (2) significantly stimulates prostaglandin production by cultured cells.

Effect of sucralfate and its components on taurocholate-induced damage to rat gastric mucosal cells in tissue culture

International Nuclear Information System (INIS)

Romano, M.; Razandi, M.; Ivey, K.J.

1990-01-01

The present study evaluated the effect of sucralfate and its components, sucrose octasulfate and aluminum hydroxide, on: (1) damage to rat cultured gastric mucosal cells induced by sodium taurocholate in a neutral environment and in conditions independent of systemic factors, (2) prostaglandin E2 and on 6-keto prostaglandin F1 alpha release by cultured cells, and (3) sulfhydryl content of cultured cells. Cell damage was quantitated by chromium-51 release assay. Prostaglandin E2 and 6-keto prostaglandin F1 alpha were measured by radioimmunoassay. Total sulfhydryl content of cultured cells was determined calorimetrically. Microscopically, sucralfate was found to adhere tightly to epithelial cell surfaces despite frequent washings. Sucralfate 2 mg/ml and 5 mg/ml significantly decreased taurocholate-induced damage, reducing taurocholate-induced specific 51Cr release by 11.8 points (equal to 29% decrease in cell damage, P less than 0.01) and 22.9 points (equal to 56% decrease in cell damage, P less than 0.001), respectively. Sucrose octasulfate and aluminum hydroxide did not exert significant protection against damage induced by sodium taurocholate. The protective effect of sucralfate was not prevented by indomethacin, nor was it counteracted by the sulfhydryl blocker, iodoacetamide. Sucralfate, but not its components, significantly and dose-dependently stimulated prostaglandin E2 (r = 0.94, P less than 0.05) and 6-keto prostaglandin F1 alpha (r = 0.89, P less than 0.05) production by cultured cells. Neither sucralfate nor its components affected sulfhydryl content of cultured cells. In conclusion, sucralfate, but not its components, (1) protects rat gastric mucosal cells against taurocholate-induced damage in conditions independent of systemic factors and in a neutral environment and (2) significantly stimulates prostaglandin production by cultured cells

A subpopulation of dopaminergic neurons co-expresses serotonin in ventral mesencephalic cultures but not after intrastriatal transplantation in a rat model of Parkinsons disease

DEFF Research Database (Denmark)

Di Santo, Stefano; Seiler, Stefanie; Ducray, Angélique

2017-01-01

Cell replacement therapy is a promising avenue into the investigation and treatment of Parkinson’s disease (PD) and in some cases significant long-term motor improvements have been demonstrated. The main source of donor tissue is the human fetal ventral mesencephalon (VM), which consists...... 30% of the dopaminergic neurons in the donor tissue co-expressed serotonin, no co-localization could be detected in grafts one month after intrastriatal transplantation into hemi-parkinsonian rats. In conclusion, a significant and susceptible sub-population of dopaminergic neurons in fetal VM tissues...... both fetal rat and human dissociated, organotypic and neurosphere VM cultures as well as an animal model of PD were investigated. In dissociated rat VM cultures approximately 30% of the TH positive neurons co-expressed serotonin, while no co-localization with GABA was observed. Interestingly, co...

Embryonic stem cells and prospects for their use in regenerative medicine approaches to motor neurone disease.

Science.gov (United States)

Christou, Y A; Moore, H D; Shaw, P J; Monk, P N

2007-10-01

Human embryonic stem cells are pluripotent cells with the potential to differentiate into any cell type in the presence of appropriate stimulatory factors and environmental cues. Their broad developmental potential has led to valuable insights into the principles of developmental and cell biology and to the proposed use of human embryonic stem cells or their differentiated progeny in regenerative medicine. This review focuses on the prospects for the use of embryonic stem cells in cell-based therapy for motor neurone disease or amyotrophic lateral sclerosis, a progressive neurodegenerative disease that specifically affects upper and lower motor neurones and leads ultimately to death from respiratory failure. Stem cell-derived motor neurones could conceivably be used to replace the degenerated cells, to provide authentic substrates for drug development and screening and for furthering our understanding of disease mechanisms. However, to reliably and accurately culture motor neurones, the complex pathways by which differentiation occurs in vivo must be understood and reiterated in vitro by embryonic stem cells. Here we discuss the need for new therapeutic strategies in the treatment of motor neurone disease, the developmental processes that result in motor neurone formation in vivo, a number of experimental approaches to motor neurone production in vitro and recent progress in the application of stem cells to the treatment and understanding of motor neurone disease.

Somatic donor cell type correlates with embryonic, but not extra-embryonic, gene expression in postimplantation cloned embryos.

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Ryutaro Hirasawa

Full Text Available The great majority of embryos generated by somatic cell nuclear transfer (SCNT display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts. The embryos retrieved from the uteri were separated into embryonic (epiblast and extraembryonic (extraembryonic ectoderm and ectoplacental cone tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs (>2-fold vs. controls than did the extraembryonic tissues (P<1.0 × 10(-26. In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1-5% per embryos transferred in our laboratory, because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.

Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds.

Science.gov (United States)

Zhou, Jin; Zhang, Ye; Lin, Qiuxia; Liu, Zhiqiang; Wang, Haibin; Duan, Cuimi; Wang, Yanmeng; Hao, Tong; Wu, Kuiwu; Wang, Changyong

2010-07-01

Embryonic stem (ES) cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy. The main catalyst for ES cells differentiation is the growth into embryoid bodies (EBs), which are utilized widely as the trigger of in vitro differentiation. In this study, a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established. When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds, they grew into aggregates gradually and formed simple EBs with circular structures. After 7 days' culture, they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers. Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types; they were also able to form into tissue-like structures. Moreover, with introduction of ascorbic acid, ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19. The results demonstrated that collagen/Matrigel scaffolds supported EBs formation and their subsequent differentiation in a single three dimensional environment. Copyright 2010 Institute of Genetics and Developmental Biology and the Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Self-organization of human embryonic stem cells on micropatterns

Science.gov (United States)

Deglincerti, Alessia; Etoc, Fred; Guerra, M. Cecilia; Martyn, Iain; Metzger, Jakob; Ruzo, Albert; Simunovic, Mijo; Yoney, Anna; Brivanlou, Ali H.; Siggia, Eric; Warmflash, Aryeh

2018-01-01

Fate allocation in the gastrulating embryo is spatially organized as cells differentiate to specialized cell types depending on their positions with respect to the body axes. There is a need for in vitro protocols that allow the study of spatial organization associated with this developmental transition. While embryoid bodies and organoids can exhibit some spatial organization of differentiated cells, these methods do not yield consistent and fully reproducible results. Here, we describe a micropatterning approach where human embryonic stem cells are confined to disk-shaped, sub-millimeter colonies. After 42 hours of BMP4 stimulation, cells form self-organized differentiation patterns in concentric radial domains, which express specific markers associated with the embryonic germ layers, reminiscent of gastrulating embryos. Our protocol takes 3 days; it uses commercial microfabricated slides (CYTOO), human laminin-521 (LN-521) as extra-cellular matrix coating, and either conditioned or chemically-defined medium (mTeSR). Differentiation patterns within individual colonies can be determined by immunofluorescence and analyzed with cellular resolution. Both the size of the micropattern and the type of medium affect the patterning outcome. The protocol is appropriate for personnel with basic stem cell culture training. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation. PMID:27735934

Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31

Science.gov (United States)

The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the subclonal cell line, PICM-31A1. P...

Metabolism of 4-nitrobiphenyl (NBP) by cultured rat urothelial cells

International Nuclear Information System (INIS)

Swaminathan, S.; Lang, D.B.; Reznikoff, C.A.

1986-01-01

The potential of rat urothelial cells to metabolize NBP was evaluated by incubating 4.3 x 10 7 viable cells with 20 μM [ 3 H]NBP in a serum free medium for 48 hours. The culture medium was examined for metabolites of NBP by extraction with ethyl acetate and subsequent chromatographic analysis. High pressure liquid chromatography of the solvent extract using a Whatman ODS-3, C-18 column in 70% methanol-water at a flow rate of 1 ml/min revealed two major peaks at retention times of approximately 8 and 13 min. Thin layer chromatography showed two regions of radioactivity at Rf values of 0.35 and 0.83, the latter corresponding with NBP. Based on the chromatographic data the metabolite with the retention time of 8.0 min in HPLC and an Rf of 0.35 in TLC has been tentatively identified as 4-acetylaminobiphenyl. Analysis of binding to proteins and nucleic acids following exposure to [ 3 H]NBP revealed a significant amount (0.03% of initially applied radioactivity) in the protein fractions. Control samples of NBP incubated in medium, without the urothelial cells revealed only the parent compound. These data suggest that rat bladder cells possess the metabolic capability to reduce NBP and to generate reactive metabolites that bind to cellular macromolecules

Efeito do Halotano sobre a Gestação e a Viabilidade Embrionária em Ratos - Estudo Experimental/Effect of Halothane on Pregnancy and Embryonic Viability in Rats - Experimental Study

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Carlos Eduardo Domingues

2012-12-01

Full Text Available Objetivo: conhecer alterações morfológicas no desenvolvimento embrionário de filhotes de ratas expostas ao halotano no período estral e gestacional e avaliar o período gestacional de maior risco. Materiais e Métodos: projeto aprovado pela Comissão de Ética e Pesquisa da UNIFENAS (Parecer Nº 11ª/2008. Foram utilizadas 25 ratas da linhagem Wistar distribuídas em cinco grupos. Grupo 1: controle (n=5 ratas com prenhez identificada sem exposição ao halotano. Grupo 2: (n=5 ratas expostas ao halotano no período estral. Grupos 3: (n=5 ratas expostas ao halotano no 8º-10º dia de prenhez. Grupo 4: (n=5 ratas expostas ao halotano no 11º a 13º dia e Grupo 5:(n=5 14º a 16º dia. Os animais foram expostos ao halotano (0,8% por 30 minutos com oxigênio a 100%. Os filhotes, analisados quanto ao tamanho, peso, alterações morfológicas e comprimento do cordão umbilical e número de nascidos vivos por grupo. Os resultados foram expressos como média (+-, desvio padrão da média, seguida de teste de Tukey. Resultados: Ocorreram alterações morfológicas no período gestacional e alterações no peso, tamanho e comprimento do cordão umbilical de filhotes do grupo 2 (3,05 ± 0,10 cm, 3,17 ± 0,17g e 2,57 ± 0,12 cm, sendo significativo (p<0,005. Conclusão: Halotano não comprometeu a fertilização dos animais estudados, mas promoveu o aparecimento de alterações morfológicas no primeiro período gestacional, mostrando o risco de teratogenicidade e consequentemente, a inviabilidade embrionária. Objective: identify morphological changes in embryonic development in rats exposed to gestational and halotano estrous period and assess the gestational period of greatest risk. Material and Methods: project approved by Commission standards and research ethics at UNIFENAS (Federal University of Alfenas (Opinion No. 11ª /2008. We used 25 female Wistar rats, distributed in five groups. Group 1: control (n=5 rats with pregnancy identified without

Molecular fingerprinting of TGFbeta-treated embryonic maxillary mesenchymal cells.

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Pisano, M M; Mukhopadhyay, P; Greene, R M

2003-11-01

The transforming growth factor-beta (TGF(beta)) family represents a class of signaling molecules that plays a central role in normal embryonic development, specifically in development of the craniofacial region. Members of this family are vital to development of the secondary palate where they regulate maxillary and palate mesenchymal cell proliferation and extracellular matrix synthesis. The function of this growth factor family is particularly critical in that perturbation of either process results in a cleft of the palate. While the cellular and phenotypic effects of TGF(beta) on embryonic craniofacial tissue have been extensively cataloged, the specific genes that function as downstream mediators of TGF(beta) in maxillary/palatal development are poorly defined. Gene expression arrays offer the ability to conduct a rapid, simultaneous assessment of hundreds to thousands of differentially expressed genes in a single study. Inasmuch as the downstream sequelae of TGF(beta) action are only partially defined, a complementary DNA (cDNA) expression array technology (Clontech's Atlas Mouse cDNA Expression Arrays), was utilized to delineate a profile of differentially expressed genes from TGF(beta)-treated primary cultures of murine embryonic maxillary mesenchymal cells. Hybridization of a membrane-based cDNA array (1178 genes) was performed with 32P-labeled cDNA probes synthesized from RNA isolated from either TGF(beta)-treated or vehicle-treated embryonic maxillary mesenchymal cells. Resultant phosphorimages were subject to AtlasImage analysis in order to determine differences in gene expression between control and TGF(beta)-treated maxillary mesenchymal cells. Of the 1178 arrayed genes, 552 (47%) demonstrated detectable levels of expression. Steady state levels of 22 genes were up-regulated, while those of 8 other genes were down-regulated, by a factor of twofold or greater in response to TGF(beta). Affected genes could be grouped into three general functional

A method for the isolation and culture of adult rat retinal pigment epithelial (RPE cells to study retinal diseases

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Janosch Peter Heller

2015-11-01

Full Text Available Diseases such as age-related macular degeneration (AMD affect the retinal pigment epithelium (RPE and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 minutes yielded 4 x 104 viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch’s membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch’s membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases.

A method for high efficiency YAC lipofection into murine embryonic stem cells.

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Lee, J T; Jaenisch, R

1996-01-01

We describe a modified protocol for introducing yeast artificial chromosomes (YACs) into murine embryonic stem (ES) cells by lipofection. With a decreased DNA:cell ratio, increased concentration of condensing agents and altered culture conditions, this protocol reduces the requirement for YAC DNA to a few micrograms, improves the recovery of neomycin-resistant ES colonies and increases the yield of clones containing both flanking vector markers and insert. These modifications enable generation of sufficient 'intact' transgenic clones for biological analysis with a single experiment. PMID:9016681

The 'ventral organs' of Pycnogonida (Arthropoda are neurogenic niches of late embryonic and post-embryonic nervous system development.

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Georg Brenneis

Full Text Available Early neurogenesis in arthropods has been in the focus of numerous studies, its cellular basis, spatio-temporal dynamics and underlying genetic network being by now comparably well characterized for representatives of chelicerates, myriapods, hexapods and crustaceans. By contrast, neurogenesis during late embryonic and/or post-embryonic development has received less attention, especially in myriapods and chelicerates. Here, we apply (i immunolabeling, (ii histology and (iii scanning electron microscopy to study post-embryonic ventral nerve cord development in Pseudopallene sp., a representative of the sea spiders (Pycnogonida, the presumable sister group of the remaining chelicerates. During early post-embryonic development, large neural stem cells give rise to additional ganglion cell material in segmentally paired invaginations in the ventral ectoderm. These ectodermal cell regions - traditionally designated as 'ventral organs' - detach from the surface into the interior and persist as apical cell clusters on the ventral ganglion side. Each cluster is a post-embryonic neurogenic niche that features a tiny central cavity and initially still houses larger neural stem cells. The cluster stays connected to the underlying ganglionic somata cortex via an anterior and a posterior cell stream. Cell proliferation remains restricted to the cluster and streams, and migration of newly produced cells along the streams seems to account for increasing ganglion cell numbers in the cortex. The pycnogonid cluster-stream-systems show striking similarities to the life-long neurogenic system of decapod crustaceans, and due to their close vicinity to glomerulus-like neuropils, we consider their possible involvement in post-embryonic (perhaps even adult replenishment of olfactory neurons - as in decapods. An instance of a potentially similar post-embryonic/adult neurogenic system in the arthropod outgroup Onychophora is discussed. Additionally, we document two

Gravity in mammalian organ development: differentiation of cultured lung and pancreas rudiments during spaceflight

Science.gov (United States)

Spooner, B. S.; Hardman, P.; Paulsen, A.

1994-01-01

Organ culture of embryonic mouse lung and pancreas rudiments has been used to investigate development and differentiation, and to assess the effects of microgravity on culture differentiation, during orbital spaceflight of the shuttle Endeavour (mission STS-54). Lung rudiments continue to grow and branch during spaceflight, an initial result that should allow future detailed study of lung morphogenesis in microgravity. Cultured embryonic pancreas undergoes characteristic exocrine acinar tissue and endocrine islet tissue differentiation during spaceflight, and in ground controls. The rudiments developing in the microgravity environment of spaceflight appear to grow larger than their ground counterparts, and they may have differentiated more rapidly than controls, as judged by exocrine zymogen granule presence.

From embryonic stem cells to functioning germ cells: science, clinical and ethical perspectives.

Science.gov (United States)

Kiatpongsan, Sorapop

2007-10-01

Embryonic stem cells have been well recognized as cells having a versatile potential to differentiate into all types of cells in the body including germ cells. There are many research studies focusing on the differentiation processes and protocols to derive various types of somatic cells from embryonic stem cells. However, germ cells have unique differentiation process and developmental pathway compared with somatic cells. Consequently, they will require different differentiation protocols and special culture techniques. More understanding and established in vitro systems for gametogenesis will greatly contribute to further progression of knowledge and technology in germ cell biology, reproductive biology and reproductive medicine. Moreover if oocytes can be efficiently produced in vitro, this will play an important role on progression in nuclear transfer and nuclear reprogramming technology. The present article will provide concise review on past important discoveries, current ongoing studies and future views of this challenging research area. An ethical perspective has also been proposed to give comprehensive summary and viewpoint for future clinical application.

Biosynthesis of the D2 cell adhesion molecule: pulse-chase studies in cultured fetal rat neuronal cells

DEFF Research Database (Denmark)

Lyles, J M; Norrild, B; Bock, E

1984-01-01

D2 is a membrane glycoprotein that is believed to function as a cell adhesion molecule (CAM) in neural cells. We have examined its biosynthesis in cultured fetal rat brain neurones. We found D2-CAM to be synthesized initially as two polypeptides: Mr 186,000 (A) and Mr 136,000 (B). With increasing...

Neuroprotective Effect of Carnosine on Primary Culture of Rat Cerebellar Cells under Oxidative Stress.

Science.gov (United States)

Lopachev, A V; Lopacheva, O M; Abaimov, D A; Koroleva, O V; Vladychenskaya, E A; Erukhimovich, A A; Fedorova, T N

2016-05-01

Dipeptide carnosine (β-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatography-mass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.

Transplantation of Porcine Hepatocytes Cultured with Polylactic Acid-O-Carboxymethylated Chitosan Nanoparticles Promotes Liver Regeneration in Acute Liver Failure Rats

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Zhong Chen

2011-01-01

Full Text Available In this study, free porcine hepatocytes suspension (Group A, porcine hepatocytes embedded in collagen gel (Group B, porcine hepatocytes cultured with PLA-O-CMC nanoparticles and embedded in collagen gel (Group C, and PLA-O-CMC nanoparticles alone (Group D were transplanted into peritoneal cavity of ALF rats, respectively. The result showed that plasma HGF levels were elevated post-transplantation with a peak at 12 hr. The rats in Group C showed highest plasma HGF levels at 2, 6, 12, 24 and 36 hr post-transplantation and lowest HGF level at 48 hr. Plasma VEGF levels were elevated at 48 hr post-transplantation with a peak at 72 hr. The rats in Group C showed highest plasma HGF levels at 48, 72, and 96 hr post-transplantation. The liver functions in Group C were recovered most rapidly. Compared with Group B, Group C had significant high liver Kiel 67 antigen labeling index (Ki-67 LI at day 1 post-HTx (P<.05. Ki-67 LI in groups B and C was higher than that in groups A and D at days 5 and 7 post-HTx. In conclusion, intraperitoneal transplantation of porcine hepatocytes cultured with PLA-O-CMC nanoparticles and embedded in collagen gel can promote significantly liver regeneration in ALF rats.

Isolation of a primate embryonic stem cell line.

OpenAIRE

Thomson, J A; Kalishman, J; Golos, T G; Durning, M; Harris, C P; Becker, R A; Hearn, J P

1995-01-01

Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. Here we report the derivation of a cloned cell line (R278.5) from a rhesus monkey blastocyst that remains undifferentiated in continuous passage for > 1 year, maintains a normal XY karyotype, and expresses the cell surface markers (alkaline phosphatase, stage-specific embryonic antigen 3, st...

Bulky PAH-DNA induced by exposure of a co-culture model of human alveolar macrophages and embryonic epithelial cells to atmospheric particulate pollution

International Nuclear Information System (INIS)

Abbas, Imane; Garcon, Guillaume; Billet, Sylvain; Shirali, Pirouz; Andre, Veronique; Le Goff, Jeremie; Sichel, Francois; Roy Saint-Georges, Francoise; Mulliez, Philippe

2012-01-01

Because of their deep penetration in human lungs, fine airborne particulate matter were described as mainly responsible for the deleterious effects of exposure to air pollution on health. Organic constituents are adsorbed on particles surface and, after inhalation, some (polycyclic aromatic hydrocarbons, PAHs) can be activated into reactive metabolites and can bind to DNA. The formation of bulky DNA adducts has been researched after exposure of mono-and co-cultures of alveolar macrophages (AM) and human embryonic human lung epithelial (L132), to fine air pollution particulate matter Air samples have been collected with cascade impactor and characterized: size distribution (92.15% 2 /g), inorganic (Fe, AI, Ca, Na, K, Mg, Pb, etc.) and organic compounds (PAHs, etc.). 32 P post-labeling method was applied to detect bulky DNA adducts in AM and L132, in mono-and co-cultures, 72 h after their exposure to atmospheric particles at their Lethals and Effects concentrations or (LC or CE) to 50% (i.e. MA: EC 50 = 74.63 μg/mL and L132: LC-5-0 = 75.36 μg/mL). Exposure to desorbed particles (MA: C1= 61.11 μg/mL and L132 : C2 = 61.71 μg/mL) and B[a]P (1 μM) were included. Bulky PAH-DNA adducts were detected in AM in mono-culture after exposure to total particles (Pt), to B[a]P and desorbed particles (Pd). Whatever the exposure, no DNA adduct was detected in L132 in mono-culture. These results are coherent with the enzymatic activities of cytochrome P450 l Al in AM and L132. Exposure of co-culture to Pt, or Pd induced bulky adducts to DNA in AM but not in L132. Exposure to B[a]P alone has altered the DNA of AM and L132, in co-culture. Exposure to Pt is closer to the environmental conditions, but conferred an exposure to amounts of genotoxic agents compared to studies using organic extracts. The formation of bulky DNA adducts was nevertheless observed in AM exposed to Pt, in mono- or co-culture, indicating that they were competent in terms of metabolic activation of PAHs. The

Cellular changes in motor neuron cell culture produced by cytotoxic cerebrospinal fluid from patients with amyotrophic lateral sclerosis.

Science.gov (United States)

Gomez-Pinedo, U; Yáñez, M; Matías-Guiu, J; Galán, L; Guerrero-Sola, A; Benito-Martin, M S; Vela, A; Arranz-Tagarro, J A; García, A G

2014-01-01

The neurotoxic effects of cerebrospinal fluid (CSF) from patients with amyotrophic lateral sclerosis (ALS) have been reported by various authors who have attributed this neurotoxicity to the glutamate in CSF-ALS. Cultures of rat embryonic cortical neurons were exposed to CSF from ALS patients during an incubation period of 24 hours. Optical microscopy was used to compare cellular changes to those elicited by exposure to 100μm glutamate, and confocal microscopy was used to evaluate immunohistochemistry for caspase-3, TNFα, and peripherin. In the culture exposed to CSF-ALS, we observed cells with nuclear fragmentation and scarce or null structural modifications to the cytoplasmic organelles or to plasma membrane maintenance. This did not occur in the culture exposed to glutamate. The culture exposed to CSF-ALS also demonstrated increases in caspase-3, TNFα, and in peripherin co-locating with caspase-3, but not with TNFα, suggesting that TNFα may play an early role in the process of apoptosis. CFS-ALS cytotoxicity is not related to glutamate. It initially affects the nucleus without altering the cytoplasmic membrane. It causes cytoplasmic apoptosis that involves an increase in caspase-3 co-located with peripherin, which is also overexpressed. Copyright © 2013 Sociedad Española de Neurología. Published by Elsevier Espana. All rights reserved.

Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells.

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Jiafei Xi

Full Text Available In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs we isolated human fetal liver stromal cells (hFLSCs from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days. Basic fibroblast growth factor (bFGF is known to play an important role in promoting self-renewal of human embryonic stem (hES cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2, and transforming growth factor β (TGF-β, thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.

Embryonic chicken transplantation is a promising model for studying the invasive behaviour of melanoma cells.

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Aparna eJayachandran

2015-02-01

Full Text Available Epithelial-to-mesenchymal transition is a hallmark event in the metastatic cascade conferring invasive ability to tumor cells. There are ongoing efforts to replicate the physiological events occurring during mobilization of tumor cells in model systems. However, few systems are able to capture these complex in vivo events. The embryonic chicken transplantation model has emerged as a useful system to assess melanoma cells including functions that are relevant to the metastatic process, namely invasion and plasticity. The chicken embryo represents an accessible and economical 3-dimensional in vivo model for investigating melanoma cell invasion as it exploits the ancestral relationship between melanoma and its precursor neural crest cells. We describe a methodology which enables the interrogation of melanoma cell motility within the developing avian embryo. This model involves the injection of melanoma cells into the neural tube of chicken embryos. Melanoma cells are labelled using fluorescent tracker dye, Vybrant DiO, then cultured as hanging drops for 24 hours to aggregate the cells. Groups of approximately 700 cells are placed into the neural tube of chicken embryos prior to the onset of neural crest migration at the hindbrain level (embryonic day 1.5 or trunk level (embryonic day 2.5. Chick embryos are reincubated and analysed after 48 hours for the location of melanoma cells using fluorescent microscopy on whole mounts and cross-sections of the embryos. Using this system, we compared the in vivo invasive behavior of epithelial-like and mesenchymal-like melanoma cells. We report that the developing embryonic microenvironment confers motile abilities to both types of melanoma cells. Hence the embryonic chicken transplantation model has potential to become a valuable tool for in vivo melanoma invasion studies. Importantly, it may provide novel insights into and reveal previously unknown mediators of the metastatic steps of invasion and

Differentiation of embryonic stem cells towards hematopoietic cells: progress and pitfalls.

Science.gov (United States)

Tian, Xinghui; Kaufman, Dan S

2008-07-01

Hematopoietic development from embryonic stem cells has been one of the most productive areas of stem cell biology. Recent studies have progressed from work with mouse to human embryonic stem cells. Strategies to produce defined blood cell populations can be used to better understand normal and abnormal hematopoiesis, as well as potentially improve the generation of hematopoietic cells with therapeutic potential. Molecular profiling, phenotypic and functional analyses have all been utilized to demonstrate that hematopoietic cells derived from embryonic stem cells most closely represent a stage of hematopoiesis that occurs at embryonic/fetal developmental stages. Generation of hematopoietic stem/progenitor cells comparable to hematopoietic stem cells found in the adult sources, such as bone marrow and cord blood, still remains challenging. However, genetic manipulation of intrinsic factors during hematopoietic differentiation has proven a suitable approach to induce adult definitive hematopoiesis from embryonic stem cells. Concrete evidence has shown that embryonic stem cells provide a powerful approach to study the early stage of hematopoiesis. Multiple hematopoietic lineages can be generated from embryonic stem cells, although most of the evidence suggests that hematopoietic development from embryonic stem cells mimics an embryonic/fetal stage of hematopoiesis.

Epigenetic control of embryonic stem cell fate