Distribution of Cell Lineage Markers in Rat Pre-Implantation Embryos and Establishment of Rat Embryonic Stem-Like Cells from the Morula.
In: Biology of Reproduction, Jg. 81 (2009-07-01), Heft 1, Number 1 Supplement 1, S. 653-653
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Zugriff:
Embryonic stem (ES) cells are characterized by pluripotency, in particular, the ability to form a germline on injection into blastocysts. In the rat, establishing ES cell lines from the blastocyst is still difficult. Thus, further improvement of this technique is greatly required. There are several papers showing how certain intrinsic biological differences among species and/or strains might affect establishing rat ES-like cell lines. Herein, we characterized preimplantation embryos of Wistar-Imamichi rats using cell lineage markers, Oct4 and Cdx2. We first performed spatiotemporal expressions of Oct4 and Cdx2 in the embryos by immunohistochemical analysis. While neither Oct4 nor Cdx2 was expressed in the nuclei at the 2-cell stage, nuclear expression of Oct4 was detected at the 4-cell stage. At the morula stage, both Oct4 and Cdx2 were expressed in all nuclei. At the blastocyst stage, Oct4 was expressed in both the inner cell mass (ICM) and the trophectoderm (TE), whereas expression of Cdx2 was only localized to the TE. These results let us establish rat ES-like cell lines from both morula and blastocyst using a combination of feeder layers (rat embryonic fibroblast [REF] for primary outgrowth and SIM mouse embryo-derived thioguanine- and ouabain-resistant [STO] cells for passaging). The rat ES-like cell lines obtained from the morula maintained expression of Oct4, whereas cell lines derived from blastocysts lost pluripotency during early passage. Thus, we further characterized morula-derived ES-like cells. Oct4 was expressed in this cell after long-term culture. Its expression was also observed even after cryogenic preservation and thawing. After EGFP transfection to the morula-derived ES-like cells, expression of Oct4 was detected in EGFP-positive cells in a long-term culture. These results demonstrate that the morula, along with a combination of feeder layers, can be used to establish rat ES-like cells. To examine the contribution of morula-derived ES-like cells within chimeric embryos, we injected these cells into blastocysts. Oct4 expression disappeared in ES-like cells within 24 h after injection into the blastocysts. This rapid loss of Oct4 was not prevented by any culture media, including rat embryo culture medium, stem cell medium, and the conditioned culture medium from ES-like cells with a feeder layer. Our present results suggest that the morula can be candidate to establish rat ES cell lines.(poster)
Titel: |
Distribution of Cell Lineage Markers in Rat Pre-Implantation Embryos and Establishment of Rat Embryonic Stem-Like Cells from the Morula.
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Autor/in / Beteiligte Person: | Sano, Chiaki ; Fukui, Emiko ; Yoshizawa, Midori ; Matsumoto, Hiromichi |
Link: | |
Zeitschrift: | Biology of Reproduction, Jg. 81 (2009-07-01), Heft 1, Number 1 Supplement 1, S. 653-653 |
Veröffentlichung: | 2009 |
Medientyp: | serialPeriodical |
ISSN: | 0006-3363 (print) ; 1529-7268 (print) |
DOI: | 10.1093/biolreprod/81.s1.653 |
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