The Harry M. Zweig Memorial Fund for Equine Research

Characteristics of Stem Cells Derived from Bone Marrow
Aspirate, Adipose Tissue and Muscle

Dr. Lisa Fortier

The application of stem cells in equine veterinary therapeutics is an emerging field. In horses, stem cells hold tremendous promise for treatment of any disease that involves cell death such as nerve injury (roarers and wobblers), arthritis, bowed tendons, and suspensory ligament tears. Stem cells are genetically defined as cells that are capable of continued self-renewal through replication and of becoming cells of specific tissue types. Typically, cells such as those from skin, are only capable of dividing a couple of times before they die, and unlike stem cells they cannot mm into cells of tissue types that are different than their origin. For example, skin cells cannot turn into nerve cells, and muscle cells cannot turn into cartilage cells. However, it is the capacity of stem cells to continuously replicate and mm into nearly any cell type in the body that makes them attractive assets for tissue regeneration.

There are an increasing number of methods/biologics being developed and utilized under the guise of "stem cell therapy", particularly to treat bowed tendons. For example, bone marrow aspirate taken from a horse's breast bone (sternum) is said to contain stem cells, and there is a company (Vet-Stem, Poway, CA) that claims to isolate stem cells out of fat taken from a horse's rump. There are also assertions that stem cells are readily isolated from muscle tissue. Although a large body of information is available on stem cells, there is a generally over-simplified view of stem cells with respect to their definitions, availability, and ability to mm into assorted tissues. The presumption by many is that any cell which sticks to a tissue culture dish is a stem cell, and no further characterizations are performed. Clinically, these simplified presumptions have lead to the relatively common procedure of injecting bone marrow aspirate into tendons and suspensory ligaments, and the increasing use of fat-derived cells, even though there is no data available concerning the absolute number of stem cells that can be obtained from either source. Further, there is no information comparing the ability of stem cells, obtained from different tissues, to mm into other tissues such as nerve, tendon or cartilage. Therefore, no objective clinical recommendations regarding the optimal source of stem cells for treatment of different injuries can be made.

The premise for this proposal is to simply answer two fundamental questions so that the best possible source of stem cells for therapeutic applications can be identified. We will ask: 1) How many stem cells can be obtained from bone marrow aspirate, fat, or muscle, and 2) Which tissue source, bone marrow, fat, or muscle, provides stem cells with the greatest capacity to mm into cartilage, bone, or nerve. We will not investigate the ability of stem cells to mm into tendon since there are no established methods to drive stem cells into tendon. More importantly, there are no molecular markers specific to tendon cells, rendering interpretation of outcome data problematical.

Stem cells obtained from bone marrow, fat, or muscle are attractive since they can be auto-transplanted, i.e. obtained from and re-inserted into the same patient. Auto-transplant-ion of cells avoids potential donor-host immune rejection and disease transmission issues. In contrast, embryonic stem cells may be subject to host-immune rejection, and although we have made tremendous progress (with Zweig funding in 2003/2004) in establishing several embryonic stem cell lines, there are not yet enough cells in our bank for experimental purposes. Therefore this proposal will focus solely, on stem cells derived from adult tissues.

The first year of studies outlined in this two year proposal are designed to characterize and quantify the number of stem cells that can be obtained from bone marrow, fat, and muscle. We anticipate that a year will be required to identify and verify molecular markers that classify cells as stem cells, and then to use the identified set of markers to quantify stem cells in our target tissues. A battery of antibodies will be tested to establish a "molecular marker profile" for each stem cell type. Drs. Flaminio, Stokol and Fortier have worked together on preliminary studies to establish the feasibility of performing these studies. At least 2 out of 5 antibodies tested in our preliminary studies was identified as a putative equine bone marrow aspirate-derived stem cell marker. In this proposal, we aim to further establish a marker profile using flow cytometry (Dr. Flaminio) with complimentary gene analysis by PCR (Dr. Fortier). Our goal is to identify at least 5 antibodies that recognize stem cells since cell surface markers can be lost/altered during culture manipulations, and having 5 antibodies to monitor will allow us to identify and tolerate such losses.

In the second year , we will set out to determine which tissue yields optimal stem cells for therapeutic application to a specific site. For example, we will determine which stem cells (bone marrow, fat, or muscle) are most capable of being directed into cartilage. It is conceivable that stem cells from different sources have limited inherent capacities for direction down specific cell lines like cartilage. We will attempt to direct stem cells (as identified and characterized in year one) down cartilage, bone, and nerve cell lines using culture medium and culture methods customized for each desired tissue type. These cell lines were chosen based on our clinical impressions of where stem cells could be highly utilized, i.e. arthritis, broken bones, and nerve loss.

These studies will provide for the first time comparative data regarding the absolute cell number and differentiation capacity of stem cells derived from various tissues. The expectation is that these studies will important information regarding the optimal source of stem cells for clinical implementation of stem cell therapy.