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PubMed2006

Quantitative Oct4 overproduction in mouse embryonic stem cells results in prolonged mesoderm commitment during hematopoietic differentiation in vitro.

The Oct4 transcription factor is essential for the self-renewal and pluripotency of embryonic stem cells (ESCs). Oct4 level also controls the fate of ESCs. We analyzed the effects of Oct4 overproduction on the hematopoietic differentiation of ESCs. Oct4 was introduced into ESCs via a bicistronic retroviral vector, and cells were selected on the basis of Oct4 production, with Oct4(+) and Oct4(2+) displaying twofold and three- to fourfold overproduction, respectively. Oct4 overproduction inhibited hematopoietic differentiation in a dose-dependent manner, after the induction of such differentiation by the formation of day 6 embryoid bodies (EB6). This effect resulted from defective EB6 formation rather than from defective hematopoietic differentiation. In contrast, when hematopoiesis was induced by the formation of blast colonies, the effects of Oct4 depended on the level of overproduction: twofold overproduction increased hematopoietic differentiation, whereas higher levels of overproduction markedly inhibited hematopoietic development. This increase or maintenance of Oct4 levels appears to alter the kinetics and pattern of mesoderm commitment, thereby modifying hemangioblast generation. These results demonstrate that Oct4 acts as a master regulator of ESC differentiation.

Animals↗
PubMed2005

Shear-controlled single-step mouse embryonic stem cell expansion and embryoid body-based differentiation.

To facilitate the exploitation of embryonic stem cells (ESCs) and ESC-derived cells, scale-up of cell production and optimization of culture conditions are necessary. Conventional ESC culture methods are impractical for large-scale cell production and lack robust microenvironmental control. We developed two stirred-suspension culture systems for the propagation of undifferentiated ESCs--microcarrier and aggregate cultures--and compared them with tissue-culture flask and Petri dish controls. ESCs cultured on glass microcarriers had population doubling times (approximately 14-17 hours) comparable to tissue-culture flask controls. ESC growth could be elicited in shear-controlled stirred-suspension culture, with population doubling times ranging between 24 and 39 hours at 100 rpm impeller speed. Upon removal of leukemia inhibitory factor, the size-controlled ESC aggregates developed into embryoid bodies (EBs) capable of multilineage differentiation. A comprehensive analysis of ESC developmental potential, including flow cytometry for Oct-4, SSEA-1, and E-cadherinprotein expression, reverse transcription-polymerase chain reaction for Flk-1, HNF3-beta, MHC, and Sox-1 gene expression, and EB differentiation analysis, demonstrated that the suspension-cultured ESCs retained the developmental potential of the starting cell population. Analysis of E-cadherin-/- and E-cadherin+/- cells using both systems provided insight into the mechanisms behind the role of cell aggregation control, which is fundamental to these observations. These cell-culture tools should prove useful for both the production of ESCs and ESC-derived cells and for investigations into adhesion, survival, and differentiation phenomena during ESC propagation and differentiation.

Animals↗
PubMed2005

The caudal-related protein cdx2 promotes trophoblast differentiation of mouse embryonic stem cells.

Besides holding great promise in clinics, embryonic stem (ES) cells represent a valuable tool for studying regulation of early developmental processes, such as cell differentiation in preimplantation embryos. The caudal-related homeobox protein Cdx2 is a transcriptional regulator essential for trophoblast lineage, functioning as early as implantation. Using an inducible system, we show that gain of Cdx2 function in ES cells triggers trophoblast-like morphological differentiation, accompanied by ploidy increase, onset of expression of trophoblast-specific markers, and loss of pluripotency-associated gene expression. These data provide an insight into the genetic network that controls lineage specification and functioning in early mammalian development.

Animals↗
PubMed2005

Defining a developmental path to neural fate by global expression profiling of mouse embryonic stem cells and adult neural stem/progenitor cells.

To understand global features of gene expression changes during in vitro neural differentiation, we carried out the microarray analysis of embryonic stem cells (ESCs), embryonal carcinoma cells, and adult neural stem/progenitor (NS) cells. Expression profiling of ESCs during differentiation in monolayer culture revealed three distinct phases: undifferentiated ESCs, primitive ectoderm-like cells, and neural progenitor cells. Principal component (PC) analysis revealed that these cells were aligned on PC1 over the course of 6 days. This PC1 represents approximately 4,000 genes, the expression of which increased with neural commitment/differentiation. Furthermore, NS cells derived from adult brain and their differentiated cells were positioned along this PC axis further away from undifferentiated ESCs than embryonic stem-derived neural progenitors. We suggest that this PC1 defines a path to neural fate, providing a scale for the degree of commitment/differentiation.

Animals↗
PubMed2005

Nephrogenic factors promote differentiation of mouse embryonic stem cells into renal epithelia.

Embryonic stem (ES) cells have been induced to differentiate in vitro into a broad spectrum of specialized cell types, including hematopoietic, pancreatic, and neuronal cell types. Such ES-derived cells can provide a valuable source of progenitor cell types. Whereas undifferentiated ES cells can become integrated into a developing kidney and contribute to tubular epithelia, the ability to generate renal precursor cells in vitro has not been reported. This study used a combination of nephrogenic growth factors to differentiate ES cells into renal epithelial cells that are capable of integrating into a developing kidney with very high efficiency. Using a combination of retinoic acid, Activin-A, and Bmp7, cultured ES cells can be induced to express markers specific for the intermediate mesoderm, from which the kidneys arise. Embryoid bodies that are cultured in the presence of nephrogenic factors can respond to inductive signals and form epithelial structures in vitro. When injected into developing kidney rudiments, treated ES cells contribute to tubular epithelia with near 100% efficiency. These methods may facilitate the large-scale culture of renal epithelial precursor cells for a variety of applications.

Activins↗
PubMed1995

[Establishment of an in vitro model system for the differentiation of synaptically coupled neurons from mouse embryonic stem cells]

The investigation of fundamental differentiation processes and toxic effects during the neurogenesis in the embryo is still difficult, since there are only a few suitable cellular model systems available so far. The isolation of neurons from early embryonic origin results in a very small amount of cells and a high number of laboratory animals is required for this purpose. Permanent tumor cell lines, able to differentiate into neuron-like cell types, are only a limited alternative for the use of primary cultures. None of the known permanent cell lines is capable to form terminally differentiated, synaptically coupled neurons in vitro. Embryonic stem (ES) cells can generate cell types of all 3 germ layers and provide a new possibility to study developmental processes on a cellular level. Nevertheless the successful differentiation of neurons from ES cells has not been shown. By using the ES cell line BLC 6 and cultivation of the cells in embryo-like aggregates (embryoid bodies), we were able to differentiate efficiently neuronal cells in vitro. The electrophysiological characterization of the BLC 6-derived neurons shows that these cells carry the complex electrical properties of postmitotic neurons and are coupled by inhibitory and excitatory synapses. The BLC 6 model probably represents the only developmental cell model suitable for studies from pluripotent stem cells to terminal differentiated neurons.

Journal Article↗
PubMed1998

[Microcinematographic studies on neurodifferentiation and neurotoxicity in vitro using mouse embryonic stem cells]

The ontogeny of the mammalian nervous system remains largely unknown, mainly because of the lack of experimental systems that will allow identification of cellular mechanism involved in neuronal maturation. Recently the succesful differentiation of neurons from embryonic stem cells of the BLC-6 cell line has been shown by our group, and these cells are able to form complex neuronal networks with typical electrophysiological characteristica including postsynaptic potentials, comparable to the situation in vivo. Further characterization of neuronal cells revealed characteristic phenotypes, equipped with various neurotransmitters, e.g. glutamate. This excitatory neurotransmitter is reported to be involved in ischemia dependent neurological disorders. As the investigation of toxic effects during neuronal maturation in the embryo is usually rather difficult, due to the limited isolation of early embryonic neurons, the ES cell line seems to be an appropriate model to study effects of neurotoxicological substances. Because of the well known effect of glutamate on dendritic outgrowth and neuronal survival, the in vitro system of embryonic stem cells was applied to study the effect of glutamate intoxication by the receptor agonist NMDA during early neuronal development using videomicroscopic time lapse studies. The results point out that the NMDA induced neurotoxicity correlates with the degree of neuronal differentiation. Despite the existence of NMDA receptors in apolar, bipolar and multipolar neurons, the latter two populations were affected more dramatically than undifferentiated apolar neurons.

Journal Article↗
PubMed2004

Anti-angiogenic effect of pyridoxal 5'-phosphate, pyridoxal and pyridoxamine on embryoid bodies derived from mouse embryonic stem cells.

We previously demonstrated anti-angiogenic activity of pyridoxal 5'-phosphate (PLP) and pyridoxal (PL) using in vitro and ex vivo angiogenesis models and anti-colon tumor effect of vitamin B(6) in mice. There is growing evidence that endothelial progenitor cells (EPCs) in circulation contribute to tumor-induced angiogenesis. It is of importance to clarify whether EPC differentiation is involved in the mechanism of anti-angiogenic effect of vitamin B(6). In this study, we investigated the effect of vitamin B(6) on angiogenesis in embryonic stem cell-derived embryoid bodies (EBs). PLP suppressed angiogenesis in EBs at >or=100 microM. Among the vitamin B(6) compounds at the concentration of 200 microM, PL, as well as PLP, was the most effective suppressor of vasculogenesis, while pyridoxine was inactive. These results were consistent with their effects in rat aorta and endothelial cell assays. Interestingly, pyridoxamine (PM), which had no effect in rat aorta and endothelial cell assays, also exerted a significant suppressive effect in this model. This study demonstrated an inhibitory effect of PM on vasculogenesis in this EB model as well as PLP and PL, and suggests that suppression of EPC differentiation is at least in part responsible for the mechanism of the anti-angiogenesis effect of vitamin B(6).

Angiogenesis Inhibitors↗
PubMed2005

Differentiation of hepatocytes from mouse embryonic stem cells and its significance.

BACKGROUND: There have been lots of important discoveries in the research on embryonic stem (ES) cells; but few studies have been involving in the differentiation and differentiation ratio of hepatic stem cells. To provide a new source of hepatocytes in hepatocytes transplantation and liver tissue engineering, we investigated the differentiation and differentiation ratio of hepatocytes in the ES cells differentiating system in a certain length of time. METHODS: BALB/c ES cells from mice were cultured for differentiation in medium without leukemia inhibitory factor (LIF). During the period of differentiation of ES cells, dual immunofluorescence was performed to analyze the expression of hepatic proteins in cytoplasm such as alpha-fetoprotein (AFP) and albumin (ALB). The count of relative hepatocyte-like cells was analyzed by flow cytometry (FCM) and the hepatic differentiation ratio was obtained. RESULTS: The expression of AFP was first determined at day 7 and that of ALB at day 13. The expressions of AFP and ALB became stronger with the extention of the differentiation time. The hepatic differentiation ratio at day 13 and 21 was 5.5% and 10.4%, respectively. CONCLUSION: ES cells could differentiate into hepatocytes, which should be a new source of hepatocytes in transplantation of hepatocytes and liver engineering.

Animals↗
PubMed2006

[The effects of embryonic bodies on differentiation of mouse embryonic stem cells to neuronal cells in vitro].

This study was directed at assessing the effects of embryonic bodies on differentiation of the embryonic stem cells. The embryonic bodies of different days gained by means of hanging, suspending and hung-suspended were processed by all-trans retinotic acids for 4 days. Then they were detected by immunocytochemistry methods and were measured to detect the fluorescence intensity changes of calcium after Glu stimulation. During this period, we observed the differentiation process and compared the differentiation ratio of embryonic stem cells processed by means of hanging, suspending for several days. It was found that the differentiation ratio of EBs obtained by suspending for 3 days, or by hanging for 3 days and suspending for 1 day, is relatively higher, which can help us find out the intrinsic differentiation mechanism of embryonic stem cells.

Animals↗
PubMed1997

Myogenesis in cultures of uniparental mouse embryonic stem cells: differing patterns of expression of myogenic regulatory factors.

The expression of myogenic regulatory factors (MRFs) in cultures of androgenetic (AG) and parthenogenetic (PG) embryonic stem (ES) cells were analyzed to identify a role for imprinted genes in the myogenic program. The time course and levels of expression of myf5, myogenin, MyoD1 and myf6 were assessed by semi-quantitative RT-PCR. A more rapid induction of myogenin expression was seen in AG ES cell cultures compared to control D3 ES cells, and myf6 was expressed by AG but not D3 cells. Persistence of myf5 and MyoD1 expression at late stages of AG cell culture suggests that proliferation and differentiation are maintained. Myogenic differentiation was delayed in PG ES cells, but abundant levels of myogenin and myf6transcripts were subsequently observed. Absence of myf5 expression and only low MyoD1 expression at later stages of culture demonstrate a decline in proliferation in PG cultures. Igf2 was induced to high levels in the late phase of both AG and D3 but not PG cell cultures, indicating paternal allele-specific expression. Igf2 expression correlated with expression of MRF genes associated with myoblast proliferation rather than terminal differentiation. H19 was expressed at very low levels in both AG and PG ES cell cultures. The delay in myogenesis in PG cultures suggests that imprinted genes other than Igf2 and H19 play a role at early stages of the myogenic program.

Animals↗
PubMed1996

Elevation of cyclic AMP levels in mouse embryonic stem cells by insulin related peptides.

The preimplantation mammalian embryo has been shown to respond to exogenous insulin-like growth factors and insulin itself, however, the most quantitatively important source of these peptides and the receptors through which they exert their effects are unclear. Whilst the type 1 insulin-like growth factor (IGF) receptor is believed to act primarily through tyrosine phosphorylation of the substrate protein alpha IRS-1, evidence for a signalling role for the type 2 receptor is disputed, some evidence pointing to mediation through G protein-dependent calcium ion flux. We have examined the response of murine embryonic stem cells, as a model for the cells of the preimplantation embryo, to IGF-I, IGF-II, insulin and analogs of IGF-II: R6 IGF-II and des (1-6) IGF-II. In response to all of these peptides, except R6 IGF-II, elevation of intracellular cyclic AMP occurs. As R6 IGF-II binds with higher affinity to the type 2 receptor than canonical IGF-II or IGF-I, and insulin fails to interact, this suggests that the elevation of cyclic AMP in response to the other insulin related peptides (IRPs) is not through the type 2 receptor. We conclude that either the type 1 receptor has a previously uncharacterized direct or indirect effect on intracellular cyclic AMP levels, or that there is a further, as yet uncharacterized, receptor active in embryonic stem cells.

Animals↗
PubMed2005

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

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

Apoptosis↗
PubMed2004

Functionally deficient neuronal differentiation of mouse embryonic neural stem cells in vitro.

Embryonic mouse neural stem cells (NSCs) were isolated from E14 mice, multiplied in medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and plated in laminin-coated wells in basic serum-free neurobasal medium. After 7 days in vitro, approximately 20% of the embryonic mouse NSCs developed into morphologically and biochemically fully maturated neurons, with extensive dendrites and multiple synaptic contacts. However, even after 22 days of culture, none of these neurons developed voltage-dependent sodium-channels characteristic for a functional neuron. Apparently, the morphological differentiation and the electrophysiological maturation of an embryonic mouse NSC into a neuron are independently regulated.

Animals↗