ES-cells

In the private and the public sectors, projects are ongoing in which random mutations are generated in murine embryonic stem cells on a large scale (ES cell libraries). Tagged and presumably inactivated genes are easily identified by sequencing. These stem cells can be used to generate the respective knockout mice. 

This suggests that cyclin A2 is not essential for the early embryonic cell cycles. Also D-type cyclins seem to be dispensable for the early mouse embryo cell cycle progression since embryonic stem (ES) cells do not express them at all before differentiation (Savatier et al 1996). We do not know, however, whether the D-type cyclins are also absent in the early embryo. These observations suggest that not only could the first cell cycles of the mouse embryo have specific modifications, but also further embryonic cell cycles are specifically modified as well. Mammalian embryonic cell cycles are probably modified often during development. Such studies could allow us to determine a profile of a minimal cell cycle in mammals which must, however, be much more complex than a simple S M phase embryonic cell cycle of amphibians or insects. 

Harper Embryonic stem (ES) cells used to be thought of as not having checkpoints, but recent work has shown that there are y irradiation inducible checkpoints in ES cells. 

The ES cells were encapsulated in the PMBV/PVA hydrogel by the same method used for the L929 cells. In general, ES cells form a cell aggregate called an embryoid body in suspension culture. However, it was observed that the encapsulated ES cells in the hydrogel did not form any embryoid body for 72 h. 

The PMBV/PVA hydrogel containing ES cells was dissociated by the addition of 0.2 M D-fructose solution after 3 days. After dissociation of the PMBV/PVA hydrogel, the recovered ES cells were cultured on gelatin-coated TCPS in the culture medium as usual, and the differentiation characters of recovered ES cells were estimated by alkaline phosphatase (ALP) staining (Fig. 14). 

ALP staining was performed to estimate the functionality of recovered ES cells, and it was found that the ES cells with undifferentiated character were well stained... 

ES cells in the PMBV/PVA hydrogel ES cells under the suspension culture... 

ES cell colony which was formed after ES cell colony and derived recovering from the PMBV/PVA hydrogel, (differentiated) cells which... 

Fig. 14 Alkaline phosphatase staining of recovered ES cells after dissociation of PMBV/PVA hydrogel (left), and of the ES cells cultured on PMB30 (right). The undifferentiated ES cells were well stained. ES cells encapsulated in the PMBV/PVA hydrogel maintained their undifferentiated character during the 3 days of encapsulation...

ES cells aggregated into embryoid bodies and propagated in growth factors, including platelet-derived growth factor, differentiate rapidly to include CNS glial cells. 

About 40% of this population express markers of oligodendrocytes, and a similar proportion have astrocytic markers. To test the potential of these cells in vitro, ES cells propagated in this fashion were transplanted into the spinal cord or cerebral ventricles of myelin-deficient rats. Two weeks after transplantation, ES derived cells were present in the dorsal columns of the spinal cord both at the implant and several millimeters in both directions from that site. The mouse origin of the ES cells transplanted into rat was confirmed by analysis of mouse satellite DNA in the grafts. Similarly, intraventricular transplanted cells formed myelin in multiple brain regions [29]. These and other results support the further study of stimulated ES cells for potential therapies in the nervous system. 

II.3f II.4f Utfl +29 kb F 7F4 0 Undifferentiated embryonic cell transcription factor 1, ES-cell oncogene 1... 

Dnmtl Maintenance (functions after replication on hemimethylated DNA to restore existing methylation patterns in vitro can methylate non-methylated DNA) associates with histone deacetylase Adult/Embryo DNMTD die in utero DNA in these embryos is hypomethylated, imprinted genes are biallelically expressed ES cells from DNMTl mice are viable and capable of de novo methylation... 

Dnmt3a De novo DNA methyltransferase Adult/Embryo DNMT3a mice die at 4 weeks ES cells from DNMT3a mice are viable and capable of de novo methylation DNMT3a embryos and ES cells exhibit demethylation of centromeric satellite repeats... 

MeCP2 Contains conserved MBD motif and a transcription repression domain Represses transcription from a methylated promoter in vitro and in vivo participates in several co-repressor complexes Sin3A/HDACl-2, NCoR/Ski, Rest/CoRest Expressed in somatic tissues and in embryonic stem (ES) cells co-localizes with heavily methylated satellite DNA in mouse cells... 

MBD2b a truncated version of MBD2a (translation stars at a second methionine codon in MBD2a) that lacks the (GR)n domain was reported to have demethylase activity with direct removal of the methyl group expressed in somatic tissues but not ES cells... 

MBD3 Contains MBD domain and a C-terminal stretch of 12 glutamic acid residues mammalian protein does not bind methylated DNA in vivo or in vitro A component of the Mi2/NuRD and SMRT/HDAC5-7 deacetylase complexes Expressed in somatic tissues and in ES cells... 

MBD4 Contains MBD domain and a repair domain (T-G mismatch glycosylase) Thymine glycosylase that binds to the product of deamination at methylated CpG sites Co-localizes with heavily methylated satellite DNA in mouse cells expressed in somatic tissues and in ES cells... 

Kaiso Contains POZ domain and three Zn-fingers binds methylated DNA in a sequence specific context via Zn-fingers Inhibits transcription in vitro and in vivo partner of pi20 cathenin when localized to the cytoplasm Cytoplasmic and nuclear expressed in somatic tissues and in ES cells... 

In a more natural situation, DNA methylation and MBD proteins are thought to be involved in the long-term maintenance of the inactive state of the gene established by other factors, rather than actively enforcing it. The prevailing evidence indicates that DNA methylation in mammalian cells usually affects genes that have already been silenced for example, retroviral transcription is shut down in ES cells two days post-infection but de novo methylation of pro-viral sequences... 

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