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The Cellular Reprogramming Laboratory works toward the understanding of the mechanisms of cellular reprogramming that governs the transformation of a somatic cell into a pluripotent one. Our main goals are to generate human isogenic pluripotent stem cells and to improve the efficiency of somatic cell nuclear transfer (SCNT). For these purposes, we use three different experimental models: embryonic stem cells (ESC), SCNT, and direct reprogramming. The pluripotency of ESC makes them candidates for a wide variety of cell and tissue therapies. In particular, we study the developmental potential of parthenogenetic ESC derived from unfertilized, chemically activated oocytes. SCNT is the best approach to reprogram somatic cells. However, its efficiency is low. A greater understanding of the mechanisms involved could lead to improved efficiency or eliminate the need for a host oocyte. Our team is researching the role of epigenetic modifications and genes that are responsible for or indicative of successful reprogramming. Lastly, direct reprogramming is a way of taking the pluripotent properties inherent in ESC and transferring them to somatic cells. The generation of an embryo for isolation of ESC remains controversial; therefore we are looking to other methods to generate pluripotent stem cells that could be of therapeutic value.
www.crl.msu.edu
The Technique
We describe the application of confocal microscopy to image live embryos without compromising their developmental potential. This application stems from our laboratory's goal of developing reliable markers of nuclear reprogramming after SCNT. The only reliable marker we have today is the derivation of healthy offspring after transfer of SCNT embryos into surrogate mothers. For any other marker to be meaningful, it has to be correlated with development to term. The approach our lab has taken is to measure different embryonic characteristics (gene expression, and cell number, and allocation at blastocyst stage) and transfer these embryos into surrogate mothers to correlate the phenotype with the development of the embryo. In order accomplish our goal, a noninvasive methodology that allows survival and development of the evaluated embryos is required. To this end, we show that confocal imaging using a spinning-disk confocal microscope allows highresolution imaging of preimplantation embryos without compromising their developmental potential. This imaging technique, coupled with fluorescent reporters of gene expression or with subcellular localized fluorescent proteins, enables us to correlate the phenotype of a given embryo with its postimplantation development.
Full developmental potential of mammalian preimplantation embryos is maintained after imaging using a spinning-disk confocal microscope, p. 741.
