The Harry M. Zweig Memorial Fund for Equine Research

The Establishment of Equine Embryonic Stem Cells

Dr. Lisa Fortier

The studies outlined in this proposal are aimed at establishing the culture conditions necessary to isolate and maintain equine embryonic stem cells. An established equine stem cell line would provide a cell source for investigating treatment options for several diseases by a multitude of researchers. Beyond the extent of this proposal, our long-term goal is to use the stem cells in cartilage development studies. Embryonic stem cells (ES cells) are derived from blastocysts, which develop about one week after fertilization of the egg. At this stage, the embryo takes the form of a shell around a cluster of cells called the inner cell mass (Figure 1). It is the inner cell mass which gives rise to the ES cells. ES cells are considered totipotent as they possess the capacity to become (differentiate or transform into) literally any cell type in the body. In contrast, mesenchymal stem cells obtained from bone marrow aspirates are considered pluripotent. Mesenchymal stem cells can differentiate into many cell types including fat, muscle and cartilage, but unlike ES cells, they cannot become all cell types, for example nerve or liver. With their amazing potential to transform into any cell type, ES cells have kindled hopes for new treatments of diseases that involve cell death (eg, Parkinson’s disease) and for tissue/organ engineering and repair for diseases affecting such tissues as cartilage, nerve, and liver.

ES cells from humans and some strains of mice have been maintained in culture for years and have remained totipotent. In addition, there are established culture systems for ES cells which can induce them to differentiate into nerve, muscle, liver, and heart muscle cells. In contrast, pig and bovine ES cells differentiate very rapidly (12-24 hours) and spontaneously into any of several cell types. This does not render the bovine or pig ES cells useless because their differentiated cell types, for example liver cells, can be maintained and studied, but they are not currently maintainable as ES cells despite intense investigation by several research groups. It may be summarized that ES cells from different species behave differently and that these differences are not understood. In fact, the exact same culture conditions used to study ES cells in one species can yield very different results in another. To our knowledge, there have been no studies performed in pursuit of equine ES cells and while failure to maintain bovine and pig to maintain ES cells may cause concern, inter-species differences may allow the establishment of successful culture condition for equine ES cell lines.

For the studies in this proposal, ES cells will be retrieved through routine uterine flushing of mares 8 days after breeding and ovulation. With hormonal supplementation, mares will typically cycle and can be bred for nine months out of the year, suggesting that our research endeavors should not be hampered by a lack of embryos. With funding from the Travers Thoroughbred Research Committee, we have gained initial experience in equine ES cell harvest and propagation. We were able to obtain eleven embryos from five mares, each flushed three times over eight weeks.

After embryos are retrieved, the inner cell mass is isolated for culture. While the cells are initially growing in culture, there are distinct cellular characteristics (morphology) which can be used to determine if the inner cell mass cells remain as totipotent ES cells, or if they have differentiated into other cell types. Because isolating the inner cell mass requires specialized equipment and expertise and because recognizing the morphologic characteristics of ES versus differentiated cell types requires a great deal of experience, our initial embryos were shipped to Dr. Neil Talbot at the USDA Gene Evaluation and Mapping Laboratory. Dr. Talbot has ten years experience in the isolation and culturing of pig and bovine ES cells and was amenable to assisting us in our initial attempts to isolate equine ES cells. Dr. Fortier traveled to Dr. Talbot’s laboratory to study the techniques of embryo dissection and ES cell culture. With the purchase of a few specialized pieces of equipment and supplies we are confident that embryo dissection and ES culture can be performed here at Cornell University. Dr. Talbot has agreed to remain as a collaborator (letter appended). Through the use of digital photography, we will be able to email photographs of the cells to Dr. Talbot for immediate consultation.

The embryo donor mares used in the previous studies and those for this study reside at the Cornell Equine Research Park and are available for use in this project (see appended letter from Carol Collyer). During the experimental period, this proposal would simply fund the mares’ board charges. The mares are not harmed from by the procedures described in this proposal. We are fortunate to have the technology and expertise for retrieval of equine embryos available through Dr. Volkmann in our Theriogenology section. Drs. Volkmann and Hanlon of the Cornell Theriogenology Section, Department of Clinical Sciences, obtained all embryos for previous studies and Dr. Volkmann will function in the same capacity for this proposal (Dr. Hanlon will no longer be at Cornell University). A substantial bonus of this project is the incidental training of Theriogenology residents and veterinary students in very relevant aspects of breeding soundness examinations, artificial insemination, and embryo retrieval and handling for potential clinical applications in embryo transfer.

Our objective is to determine the culture conditions necessary to maintain equine ES cells in a totipotent, undifferentiated state. There are infinite culture conditions that may be used, so despite the differences between species, we will draw on the experiences of others with regard to ES cell culture. Again, through the funding of the Travers Thoroughbred Research Committee, we were able to attempt multiple culture conditions in our pursuit of ES cell culture. To date, we have been successful in maintaining the cells from one embryo as ES cells for four days in culture (Figure 2). This is encouraging for the potential establishment of an equine ES cell line given the rapid (12-24 hours) differentiation of ES cells isolated from either the pig or bovine.

If successful, we will be the first to establish an equine ES cell line. These studies will undoubtedly stimulate interests in further ES cell research for potential improvements in equine health for such diseases as arthritis, bowed tendons, roarers, equine spinal cord patients such as wobblers, and liver degeneration.