The Harry M. Zweig Memorial Fund for Equine Research

The Final Verification of Equine Embryonic Stem Cell Lines

Dr. Lisa Fortier

In this proposal, our aim is to further characterize and define putative equine embryonic stem cell lines (ES cells) which we established during the Zweig 2003 funding period.  The studies outlined in this proposal will definitively identify our equine ES cell lines true totipotent embryonic stem cells, capable of becoming any cell type in the body.  An established equine stem cell line would provide a cell source for a multitude of researchers investigating treatment options for any disease that is characterized by a paucity of cells, for example tendonitis.  Beyond the extent of this proposal, our long-term goal is to not only provide other scientists with equine ES cells, but to also provide them with the knowledge necessary to propagate equine ES cells.  The long-term goal of our laboratory is use the stem cells in cartilage development studies with emphasis on cellular changes that occur as a result of aging which might lead to the development of osteoarthritis.  We have no intention of using ES cells to clone horses.

Embryonic stem cells (ES cells) are derived from blastocysts, which develop about one week after fertilization of the egg, prior to implantation of the embryo implants in the uterus.  At this stage, the embryo takes the form of a shell around a cluster of cells called the inner cell mass (ICM).  It is the ICM which gives rise to the ES cells.  ES cells are considered totipotent as they possess the capacity to become (differentiate 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, Parkinsons disease in human beings, tendonitis and arthritis in horses) and for tissue/organ engineering and repair for diseases affecting such tissues as cartilage, nerve, and liver.

The definition of an ES cell is constantly evolving.  The two key features which define ES cells are: 1) their capacity for prolonged culture (1 year duration) without differentiating into any cell type, and 2) throughout long periods of cultivation in culture, their retention of the ability to develop into any cell type in the adult body.  Several markers for ES cells have been proposed, but there are clear differences between ES cells from various species with respect to expression of these markers.  In this proposal, we aim to use several complimentary approaches to establish markers for equine ES cells and firmly establish our ES cell lines as totipotent ES cells.

In tissue culture, isolated ES cells can either spontaneously differentiate into any cell type or be propagated as ES cells for prolonged periods of time.  During the 2003 funding period, we established methods for isolation of the ICM and culture conditions necessary to support the propagation of equine ES cells.  Currently ( 8/17/03 ) our oldest culture of ES cells is 10 weeks old.  As stated above, a hallmark identification of ES cells is the capacity for prolonged (1 year) propagation in a totipotent state.  While the existence of 10-week old cultures that appear to be ES cells based on their shape (morphology) and staining for markers (immunohistochemistry) is encouraging, further propagation with continuous testing is required to definitively identify our cells as totipotent ES cells.

The goal of this proposal is to build on the progress made during the 2003 funding period where we established two putative ES cell lines.  In the studies outlined in this proposal, we will continue to culture the ES cells for a minimum of one year.  Like human ES cells, equine ES cells are best maintained on a feeder layer of cells.  The feeder cells are irradiated prior to use so that they can't divide and take over a culture, yet they can still synthesize proteins.  These cells provide unknown factors which are necessary for ES cells survival and to maintain them in a totipotent state.  We established feeder cells during the 2003 funding period and have frozen stocks available for use.  We will propagate our putative ES cells every 4-7 days onto fresh feeder cells.   During the extended propagation of ES cells, we will regularly perform (every 3 months) a series of assays to determine if our ES cell lines retain the characteristics of totipotent ES cells.  We will then be able to freeze stocks of ES cells for future use.

The series of assays to define these ES cells will begin with flow cytometry and cell sorting.  ES cells grow as colonies rather than as individual cells.  Within each colony there is the potential for a mixed cell population; some ES cells and some cells which are committed to becoming a cell type like a muscle cell.  Through flow cytometry and cell sorting we will be able to select only those cells that are ES cells for further culture.  The selected cells will be propagated and tested to ensure that they remain as stable ES cells.  We will test for expression of cell surface makers specific to embryonic stem cells through immuno-histochemistry and we will assay cells for telomerase activity.  Telomerase activity remains high in stem cells and is important for prolonged self-replication.  Non-ES cells such as cartilage or muscle cells have low telomerase activity and are only capable of 30-40 rounds of replication in contrast to stem cells which are theoretically capable of infinite replication.  Finally, we will assess chromosome number and structure of our ES cells through a process called karyotyping.  Alterations in chromosome number (normal is 64 for the horse) and in chromosome structure have been reported in human ES cells and indicate instability and deterioration of a cell line.  A successful outcome for this proposal would be propagation of equine ES cells for one year with the cells retaining normal ES cell markers, high telomerase activity and a normal karyotype.

The culture and propagation of the ES and feeder cells will be performed in the PIs laboratory.  All necessary equipment is available.  Dr. Talbot from the USDA has been invaluable for this project and has agreed to remain a collaborator (Appendix 2).  Through the use of digital photography, we will be able to email photographs of the cells to Dr. Talbot for immediate consultation.  We are fortunate to have two other experienced collaborators, Dr. Yen for flow cytometry (Department of Biomedical Sciences, Cornell University ) and Dr. Lear for equine karyotyping (The Gluck Research Center, University of Kentucky ).

If successful, 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.