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Biomedical subjects

Masako Tada

Publications and source records attributed to Masako Tada.

11 recordsLinked to original sources
PubMed2006

Targeted chromosome elimination from ES-somatic hybrid cells.

To engineer a stem cell genome, we developed a technology for targeted elimination of chromosomes from mouse embryonic stem (ES)-somatic hybrid cells. Here we demonstrate the use of a universal chromosome elimination cassette (CEC) for elimination of a single embryonic stem cell (ESC)-derived chromosome 11 or 12, and also both copies of chromosome 6, which harbor pluripotency-associated genes including Nanog. We attribute hybrid-cell pluripotency to the expression of Nanog from the reprogrammed somatic-cell nuclei.

Animals↗
PubMed2006

Epigenetic reprogramming of somatic genomes by electrofusion with embryonic stem cells.

Cell fusion is an approach for combining genetic and epigenetic information between two different types of cells. Electrofusion for generating hybrid cells between mouse embryonic stem cells and somatic cells, which is a type of nonchemically induced and nonvirus-mediated cell fusion, is introduced here as a highly effective, reproducible, and biomedically safe in vitro system. Under optimized electrofusion conditions, cells are ligned and form pearl chains between electrodes in response to AC pulse stimulation, and subsequently adjacent cytoplasmic membranes are fused by DC pulse stimulation. Hybrid cells survive as drug-resistant colonies in selection medium. Cell fusion is a technique that is applied widely in the life sciences. A recent topic of great interest in the field of stem cell research is the successful production of cloned animals via epigenetic reprogramming of somatic nuclei. Interestingly, nuclear reprogramming for conferring pluripotency on somatic nuclei also occurs via cell fusion between pluripotential stem cells and somatic cells. Furthermore, it has been shown that spontaneous cell fusion contributes to generating the intrinsic plasticity of tissue stem cells. Cell fusion technology may make important contributions to the fields of regenerative medicine and epigenetic reprogramming.

Animals↗
PubMed2006

Nuclear reprogramming of somatic nucleus hybridized with embryonic stem cells by electrofusion.

Cell fusion is a powerful tool for understanding the molecular mechanisms of epigenetic reprogramming. In hybrid cells of somatic cells and pluripotential stem cells, including embryonic stem (ES) and embryonic germ cells, somatic nuclei acquire pluripotential competence. ES and embryonic germ cells retain intrinsic trans activity to induce epigenetic reprogramming. For generating hybrid cells, we have used the technique of electrofusion. Electrofusion is a highly effective, reproducible, and biomedically safe in vitro system. For successful cell fusion, two sequential steps of electric pulse stimulation are required for the alignment (pearl chain formation) of two different types of cells between electrodes in response to alternating current stimulation and for the fusion of cytoplasmic membranes by direct current stimulation. Optimal conditions for electrofusion with a pulse generator are introduced for ES and somatic cell fusion. Topics in the field of stem cell research include the successful production of cloned animals via the epigenetic reprogramming of somatic cells and contribution of spontaneous cell fusion to generating intrinsic plasticity of tissue stem cells. Cell fusion technology may make important contributions to the fields of epigenetic reprogramming and regenerative medicine.

Animals↗
PubMed2005

Nanog expression in mouse germ cell development.

Nanog is a newly identified transcriptional factor bearing a homeodomain and expressed in pluripotential cells of preimplantation and early postimplantation embryos, and embryonic stem (ES) and embryonic germ (EG) cells. Knockout experiments indicate that Nanog functions as a key player in maintaining the pluripotency of stem cells. Importantly, Nanog expression is highly expressed in primordial germ cells (PGCs) of E11.5 and E12.5 mouse embryos. However, its temporal and spatial expression pattern and function in germ cells are largely unknown. To address these issues, whole embryos and cryosections of embryos were immunostained with anti-NANOG and anti-STELLA/PGC7 antibodies. NANOG expression, repressed in colonized PGCs of E7.25-E7.5 embryos, became detectable in migrating PGCs of E7.75-E8.0 embryos. Both male and female PGCs migrating in E9.5 and E10.5 embryos and colonizing the genital ridges of E11.5 and E12.5 embryos were positive for NANOG immunostaining, while the NANOG expression pattern differed between the sexes in the later developmental stage. In female gonadal PGCs of E13.5 and E14.5 embryos, NANOG became undetectable in germ cells positive for the synaptonemal complex-specific protein SCP3, while in male PGCs of E14.5-E16.5 embryos, the number of NANOG-positive germ cells drastically decreased during the mitotic arrest. No germ cells positive for NANOG were detectable in testes and ovaries of adult mice. Thus, in germ cell development, NANOG is expressed in proliferating germ cells, in which nuclear reprogramming is progressing.

Animals↗
PubMed2005

Pluripotential competence of cells associated with Nanog activity.

Nanog is a novel pluripotential cell-specific gene that plays a crucial role in maintaining the undifferentiated state of early postimplantation embryos and embryonic stem (ES) cells. We have explored the expression pattern and function of Nanog and a Nanog-homologue, Nanog-ps1.Nanog-ps1 was mapped on Chromosome 7 and shown to be a pseudogene. Immunocytochemical analysis in vivo showed that the NANOG protein was absent in unfertilized oocytes, and was detected in cells of morula-stage embryos, the inner cell mass of blastocysts and the epiblast of E6.5 and E7.5 embryos, but not in primordial germ cells of early postimplantation embryos. In monkey and human ES cells, NANOG expression was restricted to undifferentiated cells. Furthermore, reactivation of the somatic cell-derived Nanog was tightly linked with nuclear reprogramming induced by cell hybridization with ES cells and by nuclear transplantation into enucleated oocytes. Notably, mouse Nanog (+/-) ES cells, which produced approximately half the amount of NANOG produced by wild-type ES cells, readily differentiated to multi-lineage cells in culture medium including LIF. The labile undifferentiated state was fully rescued by constitutive expression of exogenous Nanog. Thus, the activity of Nanog is tightly correlated with an undifferentiated state of cells even in nuclear reprogrammed somatic cells. Nanog may function as a key regulator for sustaining pluripotency in a dose-dependent manner.

Amino Acid Sequence↗
PubMed2005

Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression.

The pluripotential cell-specific gene Nanog encodes a homeodomain-bearing transcription factor required for maintaining the undifferentiated state of stem cells. However, the molecular mechanisms that regulate Nanog gene expression are largely unknown. To address this important issue, we used luciferase assays to monitor the relative activities of deletion fragments from the 5'-flanking region of the gene. An adjacent pair of highly conserved Octamer- and Sox-binding sites was found to be essential for activating pluripotential state-specific gene expression. Furthermore, the 5'-end fragment encompassing the Octamer/Sox element was sufficient for inducing the proper expression of a green fluorescent protein reporter gene even in human embryonic stem (ES) cells. The potential of OCT4 and SOX2 to bind to this element was verified by electrophoretic mobility shift assays with extracts from F9 embryonal carcinoma cells and embryonic germ cells derived from embryonic day 12.5 embryos. However, in ES cell extracts, a complex of OCT4 with an undefined factor preferentially bound to the Octamer/Sox element. Thus, Nanog transcription may be regulated through an interaction between Oct4 and Sox2 or a novel pluripotential cell-specific Sox element-binding factor which is prominent in ES cells.

5' Flanking Region↗
PubMed2004

X-inactivation is stably maintained in mouse embryos deficient for histone methyl transferase G9a.

One of the two X chromosomes becomes inactivated during early development of female mammals. Recent studies demonstrate that the inactive X chromosome is rich in histone H3 methylated at Lys-9 and Lys-27, suggesting an important role for these modifications in X-inactivation. It has been shown that in the mouse Eed is required for maintenance of X-inactivation in the extraembryonic lineages. Interestingly, Eed associates with Ezh2 to form a complex possessing histone methyltransferase activity predominantly for H3 Lys-27. We previously showed that G9a is one of the histone methyltransferases specific for H3 Lys-9 and is essential for embryonic development. Here we examined X-inactivation in mouse embryos deficient for G9a. Expression of Xist, which is crucial for the initiation of X-inactivation, was properly regulated and the inactivated X chromosome was stably maintained even in the absence of G9a. These results demonstrate that G9a is not essential for X-inactivation.

Animals↗
PubMed2004

Histone code modifications on pluripotential nuclei of reprogrammed somatic cells.

Following hybridization with embryonic stem (ES) cells, somatic genomes are epigenetically reprogrammed and acquire pluripotency. This results in the transcription of somatic genome-derived tissue-specific genes upon differentiation. During nuclear reprogramming, it is expected that DNA and chromatin modifications, believed to function in cell-type-specific epigenotype memory, should be significantly modified. Indeed, current evidence indicates that acetylation and methylation of histone H3 and H4 amino termini play a major role in the regulation of gene activity through the modulation of chromatin conformation. Here, we show that the reprogrammed somatic genome of ES hybrid cells becomes hyperacetylated at H3 and H4, while lysine 4 (K4) of H3 becomes globally hyper-di- and -tri-methylated. In the Oct4 promoter region, histones H3 and H4 are acetylated and H3-K4 is highly tri-methylated on both the ES and reprogrammed somatic genomes, which correlates with gene activation and DNA demethylation. However, H3-K4 is also di- and tri-methylated in the promoter regions of Neurofilament-M (Nfm), Nfl, and Thy-1, which are all silent in both ES and hybrid cells. Thus, H3-K4 di- and tri-methylation of reprogrammed somatic genomes is independent of gene activity and represents one of the major events that occurs during somatic genome reprogramming towards a transcriptional activation-permissive state.

Acetylation↗
PubMed2003

Pluripotency of reprogrammed somatic genomes in embryonic stem hybrid cells.

Somatic nuclei can be epigenetically reprogrammed by factors present in undifferentiated embryonic stem (ES) cells. The acquisition of pluripotency by somatic genomes could render such cells a viable source of personalized cell type(s) for therapeutic application, avoiding the need for controversial therapeutic cloning. To investigate this possibility, we first determined the origin of transcripts in teratomas generated from mouse (ES x somatic cell) hybrid clones. Transcription of markers from the somatic genome demonstrated efficient in vivo differentiation down independent lineages. The induction of dopaminergic neurons by coculture with stromal PA6 feeder cells also demonstrated efficient capacity to differentiate in vitro. Hybrid clone-derived neurons expressed appropriate markers, and transcription of Pitx3 from the somatic genome was confirmed. When transplanted into mouse brains, the dopaminergic neurons were successfully integrated and expressed tyrosine hydroxylase. Thus, it should be possible to produce personalized ES-like cells with the reprogrammed somatic genomes.

Animals↗