Biomedical subjects

Eiji Mizutani

Publications and source records attributed to Eiji Mizutani.

At least 19 recordsLinked to original sources

PubMed2006

Efficient establishment of mouse embryonic stem cell lines from single blastomeres and polar bodies.

Recently, ES cell lines were established from single blastomeres taken from eight-cell embryos in mice and humans with success rates of 4% and 2%, respectively, which suggests that the method could be used in regenerative medicine to reduce ethical concerns over harm to embryos. However, those studies used other ES cells as supporting cells. Here, we report a simple and highly efficient method of establishing mouse ES cell lines from single blastomeres, in which single blastomeres are simply plated onto a feeder layer of mouse embryonic fibroblasts with modified ES cell medium. A total of 112 ES cell lines were established from two-cell (establishment rate, 50%-69%), early four-cell (28%-40%), late four-cell (22%), and eight-cell (14%-16%) stage embryos. We also successfully established 18 parthenogenetic ES cell lines from first (36%-40%) and second polar bodies (33%), the nuclei of which were reconstructed to embryos by nuclear transfer. Most cell lines examined maintained normal karyotypes and expressed markers of pluripotency, including germline transmission in chimeric mice. Our results suggest that the single cells of all early-stage embryos or polar bodies have the potential to be converted into ES cells without any special treatment.

Animals↗

PubMed2006

Successful mouse cloning of an outbred strain by trichostatin A treatment after somatic nuclear transfer.

Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, extremely low success rates have plagued this technique for a decade. Further in mice, the "clonable" strains have been limited to mainly hybrid F1 strains such as B6D2F1. Recently, we established a new efficient cloning technique using trichostatin A (TSA) which leads to a 2-5 fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. To further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4-5% success rates, which is comparable to 5-7% of B6D2F1. Thus, the TSA treatment is the first cloning technique to allow us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from hybrid strains, but also enables mouse cloning from normally "unclonable" strains.

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PubMed2006

Differentiation potential of parthenogenetic embryonic stem cells is improved by nuclear transfer.

Parthenogenesis is the process by which an oocyte develops into an embryo without being fertilized by a spermatozoon. Although such embryos lack the potential to develop to full term, they can be used to establish parthenogenetic embryonic stem (pES) cells for autologous cell therapy in females without needing to destroy normally competent embryos. Unfortunately, the capacity for further differentiation of these pES cells in vivo is very poor. In this study, we succeeded in improving the potential of pES cells using a nuclear transfer (NT) technique. The original pES cell nuclei were transferred into enucleated oocytes, and the resulting NT embryos were used to establish new NT-pES cell lines. We established 84 such lines successfully (78% from blastocysts, 12% from oocytes). All examined cell lines were positive for several ES cell markers and had a normal extent of karyotypes, except for one original pES cell line and its NT-pES cell derivatives, in which all nuclei were triploid. The DNA methylation status of the differentially methylated domain H19 and differentially methylated region IG did not change after NT. However, the in vivo and in vitro differentiation potentials of NT-pES cells were significantly (two to five times) better than the original pES cells, judged by the production of chimeric mice and by in vitro differentiation into neuronal and mesodermal cell lines. Thus, NT could be used to improve the potential of pES cells and may enhance that of otherwise poor-quality ES cells. It also offers a new tool for studying epigenetics.

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PubMed2006

Chorioallantoic placenta defects in cloned mice.

Somatic cell nuclear transfer technology has been applied to produce live clones successfully in several mammalian species, but the success rates are very low. In mice, about half of the nuclear transfer embryos undergo implantation, but very few survive to term. We undertook detailed histological analyses of placentas from cloned mouse embryos generated from cumulus cells at 10.5 dpc of pregnancy, by which stage most clones have terminated their development. At 10.5 dpc, the extraembryonic tissues displayed several defined histological patterns, each reflecting their stage of developmental arrest. The most notable abnormality was the poor development of the spongiotrophoblast layer of diploid cells. This is in contrast to the placental hyperplasia frequently observed in somatic clones at 12.5 dpc or later stages. A variety of structural abnormalities were also observed in the embryos. Both placental and embryonic defects likely contribute to the low success rate of the mouse clones.

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PubMed2006

Equivalency of nuclear transfer-derived embryonic stem cells to those derived from fertilized mouse blastocysts.

Therapeutic cloning, whereby nuclear transfer (NT) is used to generate embryonic stem cells (ESCs) from blastocysts, has been demonstrated successfully in mice and cattle. However, if NT-ESCs have abnormalities, such as those associated with the offspring produced by reproductive cloning, their scientific and medical utilities might prove limited. To evaluate the characteristics of NT-ESCs, we established more than 150 NT-ESC lines from adult somatic cells of several mouse strains. Here, we show that these NT-ESCs were able to differentiate into all functional embryonic tissues in vivo. Moreover, they were identical to blastocyst-derived ESCs in terms of their expression of pluripotency markers in the presence of tissue-dependent differentially DNA methylated regions, in DNA microarray profiles, and in high-coverage gene expression profiling. Importantly, the NT procedure did not cause irreversible damage to the nuclei. These similarities of NT-ESCs and ESCs indicate that murine therapeutic cloning by somatic cell NT can provide a reliable model for preclinical stem cell research.

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PubMed2006

Normal specification of the extraembryonic lineage after somatic nuclear transfer.

To examine the establishment and maintenance of trophectoderm (TE) lineage in somatic cloned blastocysts, the expression of Cdx2, a key molecule for specification of TE fate, was immunohistochemically examined simultaneously with Oct4 expression. Cloned mouse embryos were made by nuclear transfer using cumulus cells, tail-tip fibroblasts, and embryonic stem cells. After 96 h of culture, the rates of Oct4-expressing blastocysts were as low as 50% and 60% for cumulus and fibroblast clones, respectively. However, regardless of Oct4 expression, the majority of those cloned blastocysts (> 90%) normally expressed Cdx2. Thus, even though somatic cloned embryos have reduced potential to produce the inner cell mass lineage, the TE lineage can be established and maintained.

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PubMed2006

Injection of somatic cell cytoplasm into oocytes before intracytoplasmic sperm injection impairs full-term development and increases placental weight in mice.

This study investigated the effects on fertilized embryo development of somatic cytoplasm after its injection into intact mouse oocytes. Mature oocytes collected from female B6D2F1 mice were injected with cumulus cell cytoplasm of different volumes and from different mouse strains (B6D2F1, ICR, and C57BL/6), or with embryonic cytoplasm. After culture for 1 h, B6D2F1 sperm were injected into those oocytes by intracytoplasmic sperm injection (ICSI). The oocytes were examined for pre- and postimplantation developmental competence. Increases in the volume of the somatic cytoplasm from onefold to fourfold resulted in an impairment of blastocyst development and full-term development (28% and 7%, respectively, vs. 96% and 63%, respectively, in the control group; P < 0.01). An increase in the volume of somatic cytoplasm reduced the expression of POU5F1 (more commonly known as OCT4) in expanded blastocysts. The frequency of embryos that developed to the blastocyst stage did not differ when B6D2F1 or ICR somatic cytoplasm was injected, but injection of C57BL/6 somatic cytoplasm induced a two-cell block in embryo development. Injection of the cytoplasm from fertilized embryos did not reduce the frequency of embryos attaining full-term development. Interestingly, somatic cytoplasm significantly increased the placental weight of ICSI embryos, even the injection of onefold cytoplasm (0.20 +/- 0.02 [n = 32] vs. 0.12 +/- 0.02 in the control group [n = 87]; P < 0.01). These findings indicate that the injection of somatic cytoplasm into oocytes before ICSI causes a decrease in preimplantation development, clearly impairs full-term development, and causes placental overgrowth in fertilized embryos. To our knowledge, placental overgrowth phenotypes are only caused by interspecies hybridization and cloning, and in genetically modified mice. Here, we report for the first time that somatic cytoplasm causes abnormal placentas in fertilized embryos. This study suggests that somatic cell cytoplasmic material is one cause of the low rate of full-term development in cloned mammals.

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PubMed2006

Developmental ability of cloned embryos from neural stem cells.

The success rate is generally higher when cloning mice from embryonic stem (ES) cell nuclei than from somatic cell nuclei, suggesting that the embryonic nature or the undifferentiated state of the donor cell increases cloning efficiency. We assessed the developmental ability of cloned embryos derived from cultured neural stem cell (NSC) nuclei and compared the success rate with that of embryos cloned from other donor cells such as differentiated NSCs, cumulus cells, Sertoli cells and ES cells in the mouse. The transfer of two-cell cloned embryos derived from cultured NSC nuclei into surrogate mothers produced five live cloned mice. However, the success rate (0.5%) was higher in embryos cloned from cultured NSC nuclei than from differentiated NSCs (0%), but lower than that obtained by cloning mice from other cell nuclei (2.2-3.5%). Although the in vitro developmental potential to the two-cell stage of the cloned embryos derived from NSC nuclei (73%) was similar to that of the cloned embryos derived from other somatic cell nuclei (e.g., 85% in Sertoli cells and 75% in cumulus cells), the developmental rate to the morula-blastocyst stage was only 7%. This rate is remarkably lower than that produced from other somatic cells (e.g., 50% in Sertoli cells and 54% in cumulus cells). These results indicate that the undifferentiated state of neural cells does not enhance the cloning efficiency in mice and that the arrest point for in vitro development of cloned embryos depends on the donor cell type.

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PubMed2005

Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer.

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently, we elucidated by using round spermatids that, after nuclear transfer, treatment of zygotes with trichostatin A (TSA), an inhibitor of histone deacetylase, can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions [S. Kishigami, N. Van Thuan, T. Hikichi, H. Ohta, S. Wakayama. E. Mizutani, T. Wakayama, Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids, Dev. Biol. (2005) in press]. Here, we found that 5-50 nM TSA-treatment for 10 h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells, spleen cells, neural stem cells, and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further, we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus, our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques.

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PubMed2005

Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids.

Although round spermatid injection can be used to create progeny for males who do not produce mature sperm, the rate of successful embryogenesis after such procedures is significantly lower than that for similar procedures using mature spermatozoa. The mechanisms underlying this difference are unknown. In this study, we demonstrate that, unlike the normal paternal genome, the paternal zygotic genome derived from a round spermatid is highly remethylated before first mitosis after demethylation. Genomes from elongated spermatids exhibited an intermediate level of DNA methylation, between those of round spermatids and mature spermatozoa, suggesting that the male germ cell acquires the ability to maintain its undermethylated state in the paternal zygotic genome during this phase of spermiogenesis. In addition, treatment of zygotes with trichostatin A led to a significant reduction in DNA methylation, specifically in the spermatid-derived paternal genome, except for the pericentromeric regions enriched by trimethylation of Lys9 of histone H3. These data provide insight into epigenetic errors that may be associated with the poor development of embryos generated from immature spermatozoa.

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PubMed2005

Mice cloned by nuclear transfer from somatic and ntES cells derived from the same individuals.

The current success rate of cloned mice from adult somatic cell nuclei is very low, whereas it is relatively high for cloned mice from ES cell nuclei. In this experiment, we examined whether the success rate of cloning from somatic cells could be improved via nuclear transfer embryonic stem cells (ntES cells) established from somatic cell nuclei. We obtained 11 cloned mice and 68 ntES cell lines from the somatic cell nuclei of 7 mice, and cloned 41 mice were cloned from the ntES cell nuclei. Unexpectedly, the overall success rate of cloning from ntES cell nuclei in this series was no better than when using somatic cell nuclei. Interestingly, full-term cloned mice were produced only via ntES cells from two individuals, but not by direct nuclear transfer from the somatic cells, and vice versa. Ultimately, we were able to obtain clone mice from 6 out of 7 individuals using either somatic cells or ntES cells. Thus, although ntES cells as donor nuclei do not absolutely assure a better success rate for mouse cloning than somatic cells, to preserve and clone valuable individuals, we recommend that ntES cell lines be established. These can then be used as an unlimited source of donor nuclei for nuclear transfer, and thus complement conventional somatic cell nuclear transfer cloning approaches.

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PubMed2005

Generation of progeny from embryonic stem cells by microinsemination of male germ cells from chimeric mice.

Mice chimeric for embryonic stem (ES) cells have not always successfully produced ES-derived offspring. Here we show that the male gametes from ES cells could be selected in male chimeric mice testes by labeling donor ES cells or host blastocytes with GFP. Male GFP-expressing ES-derived germ cells occurred as colonies in the chimeric testes, where the seminiferous tubules were separated into green and non-green regions. When mature spermatozoa from green tubules were used for microinsemination, GFP-expressing offspring were efficiently obtained. Using a reverse study, we also obtained ES-derived progeny from GFP-negative ES cells in GFP-labeled host chimeras. Furthermore, we showed this approach could be accelerated by using round spermatids from the testes of 20-day-old chimeric mice. Thus, this technique allowed us to generate the ES cell-derived progeny even from the low contributed chimeric mice, which cannot produce ES-origin offspring by natural mating.

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PubMed2005

Round spermatids stained with mitotracker can be used to produce offspring more simply.

Although both intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) are used in infertility treatments, the rate of offspring achieved with ROSI is low compared with that achieved with ICSI. The difficulty in correctly selecting round spermatids from testicular cells is one of the causes of this phenomenon. We easily selected live round spermatids from testicular cells stained with 20 nM MitoTracker, which visualizes mitochondria without killing the cell. Using this method, we divided round spermatids into three groups based on the polarization of their mitochondria, and performed ROSI. The rate of successful offspring achieved with MitoTracker-stained ROSI was the same in all groups. This indicates that changes in the polarization of mitochondria in round spermatids are not directly related to the developmental capacity of subsequently fertilized embryos. Because this staining has no harmful effects on embryo development, the selection of spermatids by MitoTracker under a fluorescence microscope should be useful in research into and the treatment of infertility.

Aldehydes↗

PubMed2004

Propagation of an infertile hermaphrodite mouse lacking germ cells by using nuclear transfer and embryonic stem cell technology.

Animals generated by systematic mutagenesis and routine breeding are often infertile because they lack germ cells, and maintenance of such lines of animals has been impossible. We found a hermaphrodite infertile mouse in our colony, a genetic male with an abnormal Y chromosome lacking developing germ cells. We tried to clone this mouse by conventional nuclear transfer but without success. ES cells produced from blastocysts, which had been cloned by using somatic cell nuclear transfer (ntES cells) from this mouse, were also unable to produce offspring when injected into enucleated oocytes. Although we were able to produce two chimeric offspring using these ntES cells by tetraploid complementation, they were infertile, because they also lacked developing germ cells. However, when such ntES cells were injected into normal diploid blastocysts, many chimeric offspring were produced. One such male offspring transmitted hermaphrodite mouse genes to fertile daughters via X chromosome-bearing sperm. Thus, ntES cells were used to propagate offspring from infertile mice lacking germ cells.

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PubMed2004

Establishment of male and female nuclear transfer embryonic stem cell lines from different mouse strains and tissues.

Nuclear transfer can be used to generate embryonic stem cell lines from somatic cells, and these have great potential in regenerative medicine. However, it is still unclear whether any individual or cell type can be used to generate such lines. Here, we tested seven different male and female mouse genotypes and three cell types as sources of nuclei to determine the efficiency of establishing nuclear transfer embryonic stem cell lines. Lines were successfully established from all sources. Cumulus cell nuclei from F(1) mouse genotypes showed a significantly higher cumulative establishment rate from reconstructed oocytes than from other cells; however, there were no genotype differences in success rates from cloned blastocysts. Thus, the overall success depends on preimplantation development, and, once embryos have reached the blastocyst stage, the genotype differences disappear. All mouse genotypes that were tested demonstrated at least one cell line that subsequently contributed to germline transmission in chimeric mice, so these cell lines clearly possess the same potential as embryonic stem cells derived from fertilized embryos. Thus, nuclear transfer embryonic stem cells can be generated relatively easily from a variety of inbred mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly.

Animals↗

PubMed2004

Differential effect of recipient cytoplasm for microtubule organization and preimplantation development in rat reconstituted embryos with two-cell embryonic cell nuclear transfer.

In the present study, we examined the developmental ability of enucleated zygotes, MII oocytes, and parthenogenetically activated oocytes at pronuclear stages (parthenogenetic PNs) as recipient cytoplasm for rat embryonic cell nuclear transfer. Enucleated zygotes as recipient cytoplasm receiving two-cell nuclei allowed development to blastocysts, whereas the development of embryos reconstituted with MII oocytes and parthenogenetic PNs was arrested at the two-cell stage. Previous observations in rat two-cell embryos suggested that the distribution of microtubules is involved in two-cell arrest. Therefore, we also examined the distribution of microtubules using immunofluorescence. At the two-cell stage after nuclear transfer into enucleated zygotes, microtubules were distributed homogeneously in the cytoplasm during interphase, and normal mitotic spindles were observed in cleaving embryos from the two- to four-cell stage. In contrast, embryos reconstituted with MII oocytes and parthenogenetic PNs showed aberrant microtubule organization. In enucleated zygotes, fibrous microtubules were distributed homogeneously in the cytoplasm. In contrast, dense microtubules were localized at the subcortical area in the cytoplasm and strong immunofluorescence intensity was observed at the plasma membrane, while very weak intensity was detected in the central part of enucleated MII oocytes. In enucleated parthenogenetic PNs, high-density and fibrous microtubules were distributed in the subcortical and central areas, respectively. Pre-enucleated parthenogenetic PNs also showed lower intensity of microtubule immunofluorescence in the central cytoplasm than zygotes. In conclusion, the results of the present study showed that zygote cytoplasm is better as recipient than MII oocyte and parthenogenetic PNs for rat two-cell embryonic cell nuclear transfer to develop beyond four-cell stage. Furthermore, microtubule organization is involved in the development of reconstituted embryos to overcome the two-cell arrest.

Animals↗

PubMed2004

Determination of optimal conditions for parthenogenetic activation and subsequent development of rat oocytes in vitro.

The present study was undertaken to determine optimal conditions for parthenogenetic activation and subsequent development of rat oocytes. Oocytes from immature Wistar-Imamichi (WI) and Sprague Dawley (SD) rats were activated by electrical stimulation in combination with 6-dimethylaminopurine (6-DMAP) to assess whether different rat strains display different responses to activation treatment. Since the cleavage rates of activated oocytes were significantly higher in WI than SD strain rats, WI rats were used for the subsequent experiments to determine the effects of post-hCG time, culture duration, different activation protocols (electrical stimulation with 6-DMAP or ionomycin with 6-DMAP) and osmolarity of the activation medium on the activation and subsequent development of WI rat oocytes. For oocytes activated by electrical stimulation combined with 6-DMAP, the percentages of oocytes that were activated and that developed to blastocysts were higher when oocytes were collected at 18-20 h than at any other time points after hCG injection (16, 22-24 h). Culturing for 2-6 h before activation treatment markedly decreased the percentage of activated oocytes that developed to beyond the four-cell stage. There were no differences in the percentages of oocytes with pronuclear formation and subsequent development to the two-cell and blastocyst stages between oocytes that were activated by electrical stimulation or ionomycin, both followed by 6-DMAP treatment. Activation of oocytes by ionomycin and 6-DMAP, both in low osmolarity media (246 mOsM), markedly increased the cleavage rates and percentages of high quality blastocysts (71%). The optimal conditions determined in the present study with simplified activation protocols and high efficiency of activation and subsequent development of WI rat oocytes will be helpful for further research involving nuclear transfer in the rat.

Adenine↗

PubMed2004

Production of offspring from one-day-old oocytes stored at room temperature.

To determine the feasibility of preserving oocytes without freezing, we stored mouse oocytes in several media at different temperatures for one day. Confocal microscopy of the metaphase-II spindle in these stored oocytes revealed gross abnormalities in both the spindle and the arrangement of chromosomes. The abnormal spindles could not be rescued by transplanting the aged spindle-chromosome complex into a fresh enucleated oocyte. A diploid parthenogenetic development showed that some of the oocytes stored at room temperature could still develop into blastocysts (10-57%). However, oocytes stored in a refrigerator (5%) or incubator (0%) lost the potential almost entirely. Fertilization of room-temperature-preserved oocytes with fresh spermatozoa by ICSI or IVF resulted in, respectively, 4 and 10%, full-term births. These results suggest that when oocytes are stored at room temperature for one day, most have irreversible damage not only to their cytoplasm but also to the spindle. However, since at least a few percent of stored oocytes retained the potential for full-term development, it may be possible to overcome these problems and develop a simple method for preserving mammalian oocytes without freezing.

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