Biomedical subjects

Katsuhiro Ohkoshi

Publications and source records attributed to Katsuhiro Ohkoshi.

6 recordsLinked to original sources

PubMed2006

Gene expression profiling of mouse embryonic stem cell subpopulations.

We previously demonstrated that mouse embryonic stem (ES) cells show a wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM1) and that the level of expression is positively correlated with the pluripotency of ES cells. We also found that PECAM1-positive ES cells could be divided into two subpopulations according to the expression of stage-specific embryonic antigen (SSEA)-1. ES cells that showed both PECAM1 and SSEA-1 predominantly differentiated into epiblast after the blastocyst stage. In the present study, we performed pairwise oligo microarray analysis to characterize gene expression profiles in PECAM1-positive and -negative subpopulations of ES cells. The microarray analysis identified 2034 genes with a more than 2-fold difference in expression levels between the PECAM1-positive and -negative cells. Of these genes, 803 were more highly expressed in PECAM1-positive cells and 1231 were more highly expressed in PECAM1-negative cells. As expected, genes known to function in ES cells, such as Pou5f1(Oct3/4)and Nanog, were found to be upregulated in PECAM1-positive cells. We also isolated 23 previously uncharacterized genes. A comparison of gene expression profiles in PECAM1-positive cells that were either positive or negative for SSEA-1 expression identified only 53 genes that showed a more than 2-fold greater difference in expression levels between these subpopulations. However, many genes that are under epigenetic regulation, such as globins, Igf2, Igf2r, andH19, showed differential expression. Our results suggest that in addition to differences in gene expression profiles, epigenetic status was altered in the three cell subpopulations.

Animals↗

PubMed2005

Differences in gene expression patterns between somatic cell nuclear transfer embryos constructed with either rabbit granulosa cells or their derivatives.

Successful production of offspring by somatic cell nuclear transfer (SCNT) is affected by the nature of the donor cells used. The purpose of this study was to determine whether characteristic changes induced in donor cells by culture conditions influenced gene expression patterns in the resultant SCNT embryos. Rabbit granulosa cells (rGC) were cultured under different conditions, either with or without hCG, and the two derivative cell types obtained (named respectively cGC+ and cGC-) were used as donor cells for SCNT. There were characteristic differences between fresh rGC and the two derivative cell types: p450scc expression and progesterone secretion were both higher in cGC+ than in cGC-; expression of bmp4 and fgfr2 was decreased in cGC+ and cGC- compared with rGC; and cGC+ and cGC- cell types gained collagenIV expression. Use of fresh rGC, or cGC+ and cGC- derivative cells, did not alter either the developmental potencies of SCNT oocytes or cell numbers at the blastocyst stage. The expression patterns of four genes (bmp4, fgfr2, gata4, oct3/4) in SCNT embryos and in fertilized embryos were analyzed by quantitative RT-PCR. We found that oct3/4 was expressed in all embryos. The expression patterns of the other three genes showed considerable variation between the different types of embryo: bmp4 was found in most fertilized embryos but only some of rGC and none of cGC+ and cGC- derived SCNT embryos; fgfr2 was present in fertilized embryos but was present in some rGC and cGC- NT embryos and in all cGC+ NT embryos; gata4 was not expressed in fertilized embryos but was present in a few rGC and cGC+ NT embryos and in most cGC- NT embryos. Our results suggest that the gene expression patterns in SCNT embryos derived from granulosa donor cells are affected by characteristic changes to the cells during in vitro culture.

Animals↗

PubMed2004

Embryonic stem cells expressing both platelet endothelial cell adhesion molecule-1 and stage-specific embryonic antigen-1 differentiate predominantly into epiblast cells in a chimeric embryo.

We examined the expression of cell-surface markers on subpopulations of mouse embryonic stem (ES) cells to identify those that were associated with cells that had the highest pluripotency. Flow cytometry analysis revealed a wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) and stage-specific embryonic antigen (SSEA)-1 in ES cells. Almost all SSEA-1+ cells expressed a high level of PECAM- 1, and reversible repopulation was observed between PECAM- 1+SSEA-1+ and PECAM-1+SSEA-1- cells. The ES cells carrying the lacZ gene were sorted into three subpopulations: PECAM- 1-SSEA-1-, PECAM-1+SSEA-1-, and PECAM-1+SSEA-1+. Quantitative reverse transcription-polymerase chain reaction revealed a low level of Oct3/4 mRNA expression and an elevation in differentiation maker gene expression in PECAM-1- cells. To compare the pluripotency of these three subpopulations, a single cell from each was injected into eight-cell embryo and ES cells identified at later stages by X-gal staining. At the blastocyst stage, PECAM-1+ SSEA-1+/- cells were found to have differentiated into epiblast cells in high numbers. In contrast, PECAM- 1- cell derivatives localized in the primitive endoderm or trophectoderm. At 6.0-7.0 days post coitum, many PECAM-1+SSEA- 1+ cells were found in the epiblast, but few beta-gal+ cells were detected in any regions of embryos that were injected with cells from the other two populations. These results showed that the expression levels of PECAM-1 and SSEA-1 in ES cells correlated closely with their pluripotency and/or their ability to incorporate into the epiblast of chimeric embryos.

Animals↗

PubMed2003

Catalytic RAG1 mutants obstruct V(D)J recombination in vitro and in vivo.

To generate severe combined immunodeficient (SCID) livestocks for xenotransplantation, we have attempted to generate a SCID phenotype without gene knockout. Based on the reported mouse RAG1 mutants, we constructed the corresponding rabbit RAG1 mutants by mutagenesis of three residues within the catalytic domain: D602A, D710A, and E964A. As expected, these mutants each exhibited no catalytic activity on artificial substrates and inhibited recombination by the wild type RAG1. Moreover, replacement of the N-terminus of RAG1 with enhanced green fluorescent protein (EGFP) greatly increased protein stability, and the triple mutant RAG1 showed a twofold increase in its ability to inhibit wild type activity in vitro. We generated mice transgenic for the latter mutant to assess its effect on V(D)J recombination in vivo. Serum IgM levels in four out of seven transgenic mice were reduced to approximately 30-50% of control levels in four out of seven transgenic mice. Our results suggest that immunodeficient animals for regenerative medicine could be generated without gene knockout.

3T3 Cells↗

PubMed2003

In vitro oocyte culture and somatic cell nuclear transfer used to produce a live-born cloned goat.

The use of an in vitro culture system was examined for production of somatic cells suitable for nuclear transfer in the goat. Goat cumulus-oocyte complexes were incubated in tissue culture medium TCM-199 supplemented with 10% fetal bovine serum (FBS) for 20 h. In vitro matured (IVM) oocytes were enucleated and used as karyoplast recipients. Donor cells obtained from the anterior pituitary of an adult male were introduced into the perivitelline space of enucleated IVM oocytes and fused by an electrical pulse. Reconstituted oocytes were cultured in chemically defined medium for 9 days. Two hundred and twenty-eight oocytes (70%) were fused with donor cells. After in vitro culture, seven somatic cell nuclear transfer (SCNT) oocytes (3%) developed to the blastocyst stage. SCNT embryos were transferred to the oviducts of recipient females (four 8-cell embryos per female) or uterine horn (two blastocysts per female). One male clone (NT1) was produced at day 153 from an SCNT blastocyst and died 16 days after birth. This study demonstrates that nuclear transferred goat oocytes produced using an in vitro culture system could develop to term and that donor anterior pituitary cells have the developmental potential to produce term offspring. In this study, it suggested that the artificial control of endocrine system in domestic animal might become possible by the genetic modification to anterior pituitary cells.

Abnormalities, Multiple↗

PubMed2002

Production of transgenic chimera rabbit fetuses using somatic cell nuclear transfer.

We produced aggregate chimeric embryos between blastomeres from the somatic cell nuclear transfer (SCNT) embryos and blastomeres from normal embryos. The SCNT embryos were produced by fusing enucleated oocytes with GFP gene introduced fibroblast cells, which were derived from a day 16 fetus. GFP gene-introduced fibroblast cells were cultured and passaged four to 12 times over a period of 45-79 days before SCNT. After transferring them into pseudopregnant recipient rabbits, the 15-day postcoitus fetuses were collected. We examined the existence of the cells derived from SCNT embryos in the fetus stage of pregnancy to detect the GFP gene. Fetuses that were not collected continued to develop into newborn rabbits. Two hundred and thirty-six chimeric embryos were produced using 39 SCNT morula stage embryos, and these embryos were transferred to 11 recipient rabbits. As a result, 27 normally developed and 16 degenerated concepti were obtained. The GFP gene-positive signals were detected in one of the fetuses, two of the placentae, and two of the degenerated concepti. In this study, we found that the rabbit SCNT embryos have the ability to develop and differentiate in vivo. We also demonstrated a novel method of producing a transgenic rabbit using SCNT.

Animals↗