![]() ![]() Horses have much more genetic variability than typical inbred mouse strains, therefore equine ES cell line generation may be complicated by their inconsistent genetic background. An added problem to address is that the genetic background of the blastocyst plays an important role in the efficiency of deriving ES cell lines, as has been clearly demonstrated in the mouse. For therapeutic applications in people, another major concern is the use of non-human materials such as fetal bovine serum and mouse feeder cells to derive ES cells. Additionally, there is a risk for tumor formation if ES cells are not fully directed into a differentiated cell type prior to surgical implantation. One important disadvantage is the need for specific, complicated culture conditions to propagate and maintain ES cell lines in an undifferentiated state and the requirement for frequent monitoring of cultured cells for changes in genomic state to assure phenotypic stability. Since ES cells are somewhat immuno-privileged, this may be less of a concern in therapeutic uses. Another concern for clinical application of ES cells is the potential for allogenic immunogenicity. Routinely utilized methods for inner cell mass isolation necessitate destruction of an embryo, leading to ethical concerns across species, but especially in human ES cell research. ĭespite the tremendous potential ES cells hold for clinical benefit, a number of disadvantages need to be addressed. Both microsurgical dissection and immunosurgical dissection have been described for isolation of the equine inner cell mass. Complement is added to the antibody-labeled blastocyst, leading to destruction of trophoblastic lineage cells while the inner cell mass remains unharmed. The second method involves immunodissection using an antibody that targets trophoblast lineage cells of the blastocyst. This procedure involves mechanical dissection under microscopic guidance and manual separation of the inner cell mass from trophoblastic lineage cells. Across species, the most frequently utilized method is microsurgery. Two methods have been used to isolate the inner cell mass of the blastocyst for ES cell isolation. The absence of any data verifying in vivo pluripotency prevents definitive classification of these cells as true ES cell lines. To date, no reported equine ES cell lines have been proven to be pluripotent in any in vivo assay. ![]() During this time period, the harvested equine embryo is in a blastocyst or an expanded blastocysts stage. Embryo collection for equine ES isolation has been reported to range from 6–8 days post fertilization. There have been several reports describing the generation of ES-like cells from horses. In humans, this stage occurs at 5–6 days post fertilization, and the mouse at 3–4 days. In contrast, the methods used to characterize these cell types are similar and will be discussed together.ĮS cells are derived from the inner cell mass of the blastocyst stage embryo. The methods to derive ES and iPS cell lines are distinct from each other and will be discussed separately. ![]() A number of differences have been identified between ES and iPS cells that may have significant impact on the future clinical use of each cell type. Many iPS cell lines share gene expression patterns and epigenetic traits of ES cells however, the exact relationship between ES and iPS cells is still poorly understood. This reprogramming can be achieved using a number of techniques with varying efficiencies. Induced pluripotent cells (iPS) are somatic cells that have been reprogrammed to behave like an ES cell by artificially “turning on” expression of specific pluripotency genes. Multipotency defines cells that have the potential to form two or more differentiated tissues, but not necessarily form multiple germ layers. The mammalian embryo is totipotent up to the 16 cell stage, after which the cells of the morula begin to differentiate to defined fates. This differs from “totipotency” in that pluripotent ES cells cannot form placental tissues, and therefore ES cells cannot form a viable embryo without contributions from other cell types. Embryonic stem (ES) cells versus induced pluripotent (iPS) cellsīy definition, embryonic stem (ES) cells are pluripotent, meaning they can form tissues from all three primary germ layers (ectoderm, endoderm, and mesoderm) of the embryo. ![]()
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