Stem Cell Therapy

tem cell

Stherapy has been utilized in horses to help heal tendon, ligament and joint injuries for more than 25 years, and new uses are continually explored. These are mesenchymal stromal cells (MSCs) which are isolated from fetuses, foals or adult horses, as opposed to embryonic stem cells from embryos. The MSC stem cells can be isolated from almost any tissue but most commonly obtained from bone marrow, fat tissue and from the umbilical cord of newborn foals. The two main methods are use of the patient’s own cells (autologous cells) or cells from another horse (allogeneic cells). The advantages of using autologous cells is that they are not rejected by the patient’s immune system, and there are less regulations for use. Disadvantage of using autologous cells is that it takes 2 to 3 weeks to expand the cells prior to use, to get enough. This involves a two-step process requiring the horse to return for treatment--after initial sample collection.

This may hamper optimal treatment time since an adequate number of cells are not readily available. Allogeneic cell have the advantage of being already available--with time to select and potentially enhance cell functions prior to use.

The disadvantage is that cells from another horse are recognized by the patient’s immune system (and rejected) faster than autologous cells. Also the regulations are different; allogeneic cell product development is considered drug development

A number of veterinarians, clinics and stem cell companies treated thousands of horses with stem cells for many years. They started by treating soft tissue injuries and then progressed to treating joints. Some people also treated laminitis with stem cells. There was such an expansion of stem cell therapy in veterinary medicine that the FDA became involved, to make sure it was regulated and that this kind of therapy wasn’t being used inappropriately. Use of stem cells was put on hold, and the FDA ruled that stem cells fell into the category of drug therapy, with all the regulations for bringing a new drug to market. It has been difficult to overcome this hurdle, since it takes years and a lot of money to do this. There was very little stem cell use in horses for a number of years.

Today, most commercial strategies are focused on developing frozen allogeneic cell products. One approach is use of alloge-

By Heather Thomas ...................................................................................................................................................................................................................................................................................................................

neic stem cells from umbilical cord blood that could be basically off-the-shelf/storable and given to any horse. Two products have been approved in Europe, and trials are underway in North America to get equine products approved here. Two trials are looking at use of stem cells for treating osteoarthritis (OA) in horses. There are still a number of conditions in horses and humans that are difficult to treat successfully; we don’t have good treatments and/or the treatments are not 100% successful. Osteoarthritis is one of those conditions. Dr. Thomas Koch (Department of Biomedical Sciences, Ontario Veterinary College) has been working with stem cells for many years and says the fact we don’t have good treatments for OA is reflected by the many different ways that horses with joint pain are being managed and treated. “There are many products being used for OA, and veterinarians have different preferences in treatment. This indicates that no one treatment has been shown to be superior. There is interesting data in humans and animals, however, showing that stem cells may be useful in treating some joint conditions,” he says. Koch is founder and CEO of eQcell, a company that is currently starting two trials using stem cells in equine synovitis and early stages of osteoarthritis--one at University of Guelph’s Ontario Veterinary College Equine Sports Medicine and Reproductive Centre, and the other trial at the University of California-Davis Veterinary Institute for Regenerative Cures (VIRC). The Canadian study in equine fetlock and carpal joint osteoarthritis is authorized by Health Canada’s Veterinary Drug Directorate and is the first stem cell trial in Canada for treatment of equine osteoarthritis. The U.S. study in fetlock osteoarthritis is being conducted under VIRC’s Investigational New Animal Drug (INAD) with the FDA. Stem cell therapy holds a lot of promise because it has several advantages over traditional pain medications like bute (which merely mask pain and has a narrow window of safety and can have damaging side effects) that can’t be legally used in competition. There are biologic products that can be injected into joints, like PRP (platelet rich plasma) or IRAP (interleukin receptor antagonist protein), and there are fewer regulations regarding their use (as opposed to stem cells). “But it’s hit or miss whether they work, depending on the individual horse,” says Koch. Steroids have also been used for treating OA, but there is controversy about using those in joints because they may damage the cartilage. Stem cells therapies are gaining interest because they have several advantages. “There are now two cell products approved in Europe, for treating inflammatory joint pain in horses caused by synovitis and early OA. These are HorStem and Arti-Cell Forte. They both use culture expanded MSCs. HorStem utilizes cells from equine placental umbilical cord tissue, and Arti-Cell Forte isolates MSCs from peripheral blood of adult horses. These two products are both approved for use in horses with joint pain due to early stage synovitis but are slightly different in formulation,” he says. “HorStem contains only the cells (15 million stem cells) and Arti-Cell Forte is a combination product. It actually has only

By Heather Thomas ...................................................................................................................................................................................................................................................................................................................

2 million cells, plus PRP (platelet rich plasma). So it’s difficult to know if the healing effect is due to the cells or the PRP, or whether the two may have a synergistic effect working together,” says Koch.

“A study at Cornell University investigated mesenchymal stem cells from horses in their research trial, in which they damaged the cartilage in joints of research horses. Some of those joints got stem cells and some got just the injection fluid without cells. In the follow-up, the researchers saw that the cartilage in the joints that received the cell formulation were much more preserved than the joints that did not get those cells.”

There was less cartilage damage in the joints that received the cells. “I’m not sure if these therapies can turn a damaged joint into a less-damaged joint, but the Cornell study indicates that maybe we can arrest the damage and prevent further damage.”

It is exciting that two products in Europe have been approved. “One of those companies was acquired by Boehringer Ingelheim (a large international pharmaceutical company) last summer. This is a boost, to know that this big company thinks stem cells and regenerative medicine have a role to play in veterinary medicine,” says Koch. “There’s possibility now for more consistent disease management—with joint injections to keep a horse comfortable. It might only have to be every 6 to 8 months or even longer. It might depend on the horse, but for horses that do respond, we may be able to manage their disease more consistently and have some degree of joint disease arrest,” he says. This could be career-extending, for some horses.

“Our company, eQcell, is trying to do something similar with products currently available. Our trial at UC-Davis is being led by Dr. Larry Galuppo. The trial at Ontario Veterinary College in Canada is led by Dr. Judith Koenig (Equine Sports Medicine & Reproduction Centre). Both trials have approval—from Health Canada and from the FDA—since stem cells are regulated because they are considered drugs,” says Koch.

This is the reason there are no approved stem cell products currently available in North America. Other regenerative therapies like IRAP and PRP fall under the FDA’s category of devises rather than drugs. “There is a big difference between getting a device approved by FDA and getting a drug approved. It is very expensive and time-consuming to get a drug approved, which is a big impediment for veterinary medicine. From a pharmaceutical perspective it is a very small market,” he explains.

HOW THE CELLS ARE HARVESTED AND GROWN - “The cells we are working with are from umbilical cord blood of newborn foals. My PhD work reported on the presence of these cells in 2007. This had been reported in humans, but we were the first to see if we could find similar cells in equine umbilical cord blood,” he says.

“I finished my PhD and continued to work on these cells. Our company gets the cells from umbilical cord blood. Most people get it from bone marrow or fat tissue. In Europe, one product (HorStem) contains cells from the umbilical cord tissue (not blood) and the other product (Arti-Cell Forte) contains cells from the peripheral blood of adult horses--isolating cells from the blood, using my initial protocol for umbilical cord blood, since these also work for adult blood,” he explains.

“These cells adhere to certain types of plastics. In the lab we use plastic culture dishes. In the first few days a lot of cells are just floating in the media and don’t attach, and then some start attaching to the plastic. When we aspirate the media off and replace it with fresh media, this gets rid of all the floating cells. We end up with a cell population stuck to the plastic,” says Koch.

After 8 to 12 days some of the cells on the dish start to form colonies, undergo cell division and expand. “We use enzymes to lift them off the plastic; the enzymes cleave the binding without damaging the cells. We harvest them this way and split them into 3 to 5 flasks of the same size. Those cells reattach and keep growing. We can eventually grow them into billions of cells,” he says.

The original cells from umbilical cord blood come from breeding farms. “I work with Standardbred and Thoroughbred farms in southern Ontario. During foaling season they have eQcell’s collection kits, and whenever a foal is born, the attendant clamps the umbilical cord. When the foal stands up, the cord simply breaks at its natural breaking point. With the clamp across it (toward the mare side) the blood is not gushing out from the placenta, and is saved.”

The attendants use a blood transfusion bag to collect it. “These bags come preloaded from the company, with anticoagulant and a needle on the end. The attendants clean the cord and put the needle into the blood vessel within it, and the blood can drain into this collection bag. It’s non-invasive; you are just saving the blood that would otherwise drain out and be lost.” The infusion bag can be stored in a refrigerator overnight, then shipped by FedEx to the lab.

“What’s nice about this source of cells is that they are as young as we can get them, and consistent. The cells are from newborn foals, and mares that have no signs of disease during the pregnancy,” he says.

There are advantages to having very young cells. “If you get cells from fat tissue, blood or bone marrow from adult horses, they may be damaged. All cells age, so even if you have a healthy, normal animal, there are stringent requirements for donor testing.” With foal cells, there is some variability between one foal and another, but these cells are more consistent.

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PHOTOS

First page top to bottom | Photo credit: Dr. Erin Roberts, University of Calgary - Cells are stained to show the cell nucleus in turquoise. | Cells are stained to show the cell nucleus in turquoise. | Equine Cord Blood Mesenchymal Stem Cells grown on Microcarriers from PBS Biotech. Cells are stained to show the cell nucleus in green. Second page top to bottom | Photo credit: Karen Mantel, Ontario Veterinary College, University of Guelph Equine mesenchymal stomal cells being inspected during laboratory expansion. | Sedating horse prior to treatment with equine mesenchymal stomal cells. | Culture media change on equine mesenchymal stomal cells during laboratory expansion. Second page top to bottom | Dr. Thomas Koch and Horse | Lameness evaluation of horse (carpal joint flexion test).