RAT EMBRYONIC STEM-LIKE (ES-LIKE) CELLS CAN CONTRIBUTE TO MULTIPLE EARLY EMBRYO LINEAGES IN VIVO

Abstract

The rat is an important disease-based model used extensively in biomedical research. For many human diseases, including cardiovascular diseases and diabetes, the rat is a more appropriate animal model than the mouse for deciphering mechanisms underlying both normal and pathological function, not only due to the similarity of its physiology to that of humans, but also simply due to its larger size, facilitating experimental manipulation. Unfortunately, at present, no genuine rat embryonic stem (ES) cells are available, as judged by the capacity to contribute to all tissues as well as to the germline of chimeras. Therefore, in sharp contrast to the mouse, the use of gene-targeting technology in the rat via chimera generation is not yet possible. Indeed, no in vivo contribution of rat ES-like cell lines to any developing or adult tissues has been reported following blastocyst injection. Recently, for the first time, we have shown that one of our rat ES-like cell lines, ICM17, can contribute to the extraembryonic tissues of developing fetuses by microsatellite analysis. However, this PCR-based approach does not allow us to identify which specific cell types are actual descendants of the ESlike cells. The reasons underlying the apparent incapacity of rat ES-like cells to maintain the ability to contribute to developing tissues of the embryo proper are unclear. We hypothesized that these rat ES-like cells, previously derived and characterized in our lab, possess an intrinsic propensity to contribute exclusively to extraembryonic tissues, and that this in vivo bias was a reflection of the phenotype observed in vitro. Using a transgenic rat ES-like cell line expressing fluorescent proteins of the GFP family, we sought to determine in finer detail the extent to which our cells were able to contribute to the different lineages of the early embryo. Analysis of late midgestation fetuses by PCR on genomic DNA for detection of the transgene shows that the transgenic rat ES-like cells contribute exclusively to extraembryonic tissues of fetuses, confirming the results obtained by microsatellite analysis. In addition, the results obtained by immunohistochemistry on chimeric extraembryonic tissues reveal that the rat ES-like cells injected into blastocysts are able to contribute specifically to tissues of the visceral yolk sac endoderm, parietal yolk sac endoderm, as well as trophoblast giant cell lineages, clearly demonstrating the multipotency of the rat ES-like cells. Furthermore, RT-PCR and immunofluorescence analysis of the rat ES-like cultures reveals that in addition to expressing molecular markers of the undifferentiated state (Oct4), markers of primitive endoderm (Gata4/6), visceral endoderm (Foxa2), and parietal endoderm (Sparc) are also expressed in vitro. Taken together, these results suggest that although the rat ES-like cells do not contribute to tissues of the embryo proper, they are sufficiently multipotent to contribute to multiple lineages of the late midgestation rat embryo. Also, it appears that contribution to these lineages can be foreshadowed by the detection of particular lineage markers expressed in the original cultures. This research therefore represents a step forward in the search for bona fide rat ES cells. Supported by a Natural Sciences and Engineering Research Council of Canada Industrial Postgraduate Scholarship to S.-P. Demers and by Clonagen inc. (platform)