Mesenchymal stem cells in veterinary medicine

What are mesenchymal stem cells?

A stem cell is an unspecialized cell capable of self renewal and able to differentiate into a specialized cell. Embryonic stem cells are derived from the inner cell mass of early stage embryos whereas adult stem cells are found in all tissues of the body. Embryonic stem cells possess the ability to differentiate into any cell type of the body, whereas adult stem cells are partially committed towards particular cell or tissue types. From a functional standpoint, adult stem cells are divided into two categories: haematopoietic (HSC) and mesenchymal stem cells (MSC). HSCs are found in bone marrow and give rise to all blood cells. MSCs derive from the connective tissue lineage, the mesenchyme, and are found in numerous connective tissues throughout the body (Raff, 2003). MSCs are attractive in clinical therapeutics because of their ability to grow in tissue culture, differentiate into connective tissue cell types, provide growth factor support and modulate the immune response, especially providing anti-inflammatory effects (Caplan and Dennis, 2006). Adult MSCs and their therapeutic capabilities in a veterinary clinical setting are the focus of this article.

Cell secretions

MSCs secrete cytokines that are signalling proteins capable of modulating the activity of surrounding cells. Cytokines play a key role in controlling tissue-specific repair processes. The cytokines secreted by MSCs enhance the cellular function of host cells, recruit immune and repair cells from other parts of the body, and exhibit an immunomodulatory effect that reduces inflammation and controls autoimmune responses (Caplan and Dennis, 2006). MSCs secrete growth factors such as: vascular endothelial growth factor (VEGF), which promotes new blood vessel formation via angiogenesis; hepatocyte growth factor (HGF), which is responsible for tissue regeneration and angiogenesis; insulin-like growth factor (IGF) responsible for cell proliferation and inhibiting cell death; and transforming growth factor (TGF), which inhibits the activity of T-cells, B-cells, and natural killer cells and consequently dampens the immune response to injury and inflammation (Iyer and Rojas, 2008).

Tissue regeneration

The use of MSCs to regenerate joint tissue has been demonstrated in animal models in numerous publications (see below) including large animals and companion animals.

The regeneration of traumatized tissues has been demonstrated in induced meniscus and cruciate injuries using both bone marrow and periosteum derived MSCs in caprine and rat (Murphy et al, 2003;Kanaya et al, 2007). Hyaline cartilage regeneration has not only been established in rabbits, goats and pigs, but has been demonstrated with magnetic resonance imaging (MRI) in people suffering from OA (Wakitani et al, 1994; Masuoka et al, 2006; Centeno et al, 2008). Bone marrow and adipose-derived MSCs have both been shown to regenerate bone in surgically induced injury in canines and rabbits (Bruder et al, 1998; Arinzeh et al, 2003; Cui et al, 2007; Di Bella et al, 2008). However, in all animal models of induced diseases, the extent of injury does not wholly reflect the real disease processes. This emphasizes the need for comprehensive data from real clinical cases treated with MSCs.

Current veterinary clinical applications of stem cells

Published literature on the clinical use of stem cells is limited to a small number of studies in canine and equine musculoskeletal conditions.

There is only one double blind study on the clinical use of stem cells in animals, a multicentre trial in the treatment of canine OA of the coxofemoral joint with adipose-derived MSCs. In this study nine dogs were treated with MSCs and nine dogs received placebo treatment. A significant difference was seen between treated and control dogs in lameness at walk, lameness at trot, pain on manipulation and range of motion at all follow-up time points of 30, 60 and 90 days post treatment (Black et al, 2007). A further open study detailing the same treatment with adipose-derived MSCs in canine elbow OA demonstrated a significant difference in lameness at walk, lameness at trot, functional disability and joint stiffness in the treated dogs post treatment. This study did not have a control group, however, the follow-up period continued to 180 days (Black et al, 2008).

Equine joint, tendon and ligament injuries have been treated with either bone marrow or adipose-derived MSCs for the last 10 years. A double blind study of collagenase-induced tendonitis in eight horses reported positive histological changes in the group treated with adipose-derived cells in comparison to the control group. Significant improvement in fiber architecture, density and alignment was reported in the treated group as well as increased levels of an important protein for healthy tendon development; cartilage oligomeric matrix protein. However, ultrasonography performed at 6 weeks post treatment showed no changes between the treated and control groups in the rate or quality of the tendon repair (Nixon et al, 2008). Frisbie et al (2009) reported no significant difference between the use of adipose-derived and bone marrow-derived MSCs in the treatment of induced OA in the middle carpal joint of horses (Frisbie et al, 2009). Smith and Webbon report the use of culture-expanded bone marrow-derived MSCs in over 100 horses with superficial digital flexor tendinopathy. This technique has shown rapid in-filling of the tendon lesion with no negative side effects (Smith and Webbon, 2005). Del Bue et al (2008) report the treatment of tendonitis in 16 horses with adipose-derived stem cells in conjunction with platelet-rich plasma; 14 of the 16 horses treated returned to their full pre-injury function (Del Bue et al, 2008). All of these examples illustrate the promising results observed with the therapeutic use of MSCs for a variety of veterinary diseases and conditions.

Sources of stem cells

In veterinary medicine MSCs are normally derived from one of three sources: adipose tissue; bone marrow; and cord blood. Despite stem cells being found in numerous tissue types, the aforementioned are desired due to accessibility, proliferation rates in cell culture and therapeutic cell numbers. The relative high numbers of MSCs and relative ease of tissue harvest makes adipose tissue a particularly advantageous tissue type in comparison to bone marrow and cord blood (Fraser et al, 2006). This article will, therefore, focus on adipose-derived MSCs.

Availability of stem cell procedures

There are a number of challenges that a veterinary clinic faces before being able to provide stem cell therapies. First, the isolation of the stem cells can be difficult without expertise or training. Second, there are patents in the area such that a license to use certain stem cell technology is required.

Culturing cells harvested from bone marrow is complex and requires exceptionally clean laboratory facilities, a CO₂ incubator and experienced technicians to perform cell culture. Processing adipose tissue is substantially easier as the cell culture step is eliminated. However, the process used to isolate cells from fat requires training and specific laboratory equipment. The alternative is to send the excised adipose tissue to a central processing facility. Shipment of adipose tissue may compromise cell number and viability due to transportation time.

There are several companies worldwide that offer commercial stem cell treatments:

Vet-Stem is a company that offers processing of adipose tissue for both canines and equines. Vet-Stem has a license from the University of Pittsburgh for a patent that covers a form of isolated adipose-derived stem cells. Since 2003, Vet-Stem has treated over 3000 horses and more than 1250 dogs. They have a central processing facility in San Diego to which accredited veterinarians around the US ship harvested fat. This fat is then processed to isolate the stromal vascular cells and shipped back to the veterinarian who then injects the mixture into the injured site.

Regeneus has a procedure that is different to Vet-Stem and outside the Pittsburgh patent. Regeneus has treated dogs suffering from OA in Australia and New Zealand and has recently begun treating horses with tendon, ligament and joint complaints. Regeneus provides a license and training to veterinarians and nurses so that the AdiCell procedure can be performed in-house with immediate re-implantation of cells ensuring cell viability (Box 1).

VetCell is a UK-based company offering commercial bone marrow culture for treatment of tendon and ligament injuries in horses. They also offer a foal umbilical cord blood cell separation and storage facility. To the authors' knowledge there is currently no commercial adipose-derived stem cell product or processing facility in the UK.

Case selection

OA in all synovial joints can be treated with MSCs. Treatment of the spine is not possible because of the limited intra-articular space. Suitable candidates for treatment usually fall into three categories: dogs with misaligned anatomy; dogs that have already undergone corrective surgical treatments but have ongoing or progressive arthritic symptoms; and elderly animals or animals with many OA affected joints.

Clinical outcome

Adipose-derived stem cell treatment can show dramatic results in the short term; within the first month a rapid improvement is often seen in the treated canines with increased mobility and functional ability and decreased signs of pain and lameness (Black et al, 2007; Black et al, 2008). This rapid improvement is due to the anti-inflammatory effect of the MSCs. The treatment is long lasting with the earliest patients still showing sustained improvement at 2 1/2 years post initial treatment (Webster et al, unpublished data).

However, not all patients respond to the treatment with veterinarians using the therapy reporting that approximately 15% of patients show no or limited improvement on clinical examination. To date it is not understood why some patients do not respond.

Safety

Adipose-derived MSCs have been used to treat OA in canines and equines without major reported complications for over 3 years. When cells are transplanted with minimal processing and no cell culture is performed the potential risks or detrimental effects that have been associated with culturing cells are eliminated. Longer-term safety can also be assumed from the safe long-term use of other similar autologous cell transfers, such as bone marrow transplantations, that have been performed since the 1960s in human patients. Fat transfer procedures have also been safely used in human cosmetic and reconstructive surgery for many years (Hang-Fu et al, 1995).

Adverse events

There are some mild acute effects seen post MSC procedure. These are usually limited to the fat harvest surgical site swelling, bruising or seroma formation. Temporary worsening of lameness and discomfort has been reported by treating veterinarians, which is usually due to the manipulation of joints while the dog is under anaesthetic or the arthrocentesis. Commonly this is resolved within the first few days and treated dogs are generally discharged from hospital the day following treatment. Significant complications of MSC procedures have not been identified to date.

Conclusions

The last two decades has seen a surge in the research and clinical availability of stem cell treatments. The use of adult stem cells in veterinary medicine shows great promise and is likely to show rapid uptake, as commercially available safe treatments with adipose and bone marrow-derived cells become more widespread.

Adipose is an ideal tissue as MSC cell numbers are significantly higher requiring no in vitro culturing, and harvest from companion animals is routine during surgical procedures in the veterinary setting.

The AdiCell™ procedure

In 2008 a procedure called AdiCell using autologous adipose-derived cells was developed and patented to treat canine osteoarthritis (OA). The technique isolates the high numbers of MSCs found in adipose tissue along with other nucleated cell types associated with the capillaries in fat. Together, these cells make up the stromal vascular fraction. A key feature of AdiCell is that the fat is processed onsite at the veterinary clinic and the therapeutic is administered to the animal less than 1 hour after the adipose tissue has been harvested. This ensures the highest possible number of fresh viable cells are used for the therapy. Veterinary clinics are provided with the equipment required to perform the treatment, including a laminar flow cabinet, centrifuge and water-bath.

A sample of fat, approximately 20–40 g, is removed from the inguinal fat pad in dogs and the tail base in horses. Under sterile conditions in a laminar flow hood, the fat is washed in sterile saline and homogenized until the fat is approximately in 1mm³ pieces. After the addition of an enzymatic reagent, the mixture is subsequently incubated at 37°C for 20–30 minutes. The enzyme breaks down the connective tissue releasing single cells from the fat. The sample is then filtered removing any large particles of un-digested fat and centrifuged multiple times to wash the cells. The cells are re-suspended to a volume required to inject into the arthritic tendon, ligament or joint/s. The volume for each site varies depending on the site being treated. The volume ranges from 0.5 ml for the tarsus and carpus to 3 ml for the stifle, hip and elbow in the dog and 1–5 ml in the horse, depending on the size of the tendon or ligament lesion or the joint being treated. While the dog is anaesthetized the veterinarian will identify the joint/s by arthrocentesis and inject the cells. Multiple joints are treated simultaneously. In equines a tendon or ligament lesion is identified by ultrasound.

The veterinary nurse's role in AdiCell

The veterinary nurse is responsible for the tissue processing which is a specialized process. If the procedure is not performed correctly the cell number, cell viability, sterility of the sample and treatment outcome may be compromised. Timing is paramount and within the boundaries of the protocol the nurse is responsible for assessing the sample at different time points and ensuring the procedure is completed within the defined time frame.

The other critical role of the veterinary nurse is to assist with the intra-articular injections. Having a good understanding of the anatomy allows effective manipulation of the joints ensuring the veterinarian can more easily perform the arthrocentesis, which is often challenging in a severely arthritic joint.

As stem cell technologies in the veterinary setting advance, there will be a requirement for a person with both laboratory and veterinary expertise. This role is likely to be filled by the veterinary nurse and offers an exciting opportunity to broaden skills and be involved in cutting edge technologies.

These two aspects of adipose tissue provide the veterinary practitioners with the ability to perform stem cell treatments in-house within 2 hours. Furthermore, a mixed cell population, the stromal vascular fraction, is derived containing MSCs as well as T-regulatory cells, hematopoietic cells, macrophages and endothelial progenitor cells. (Varma et al, 2007) It is not yet known which of the stromal vascular fraction components are therapeutic. But a therapeutic effect, provided by a mixed cell population that MSCs alone cannot supply, has been reported. (McIntosh et al, 2006).

Due to the relatively recent emergence of adult stem cell treatments, there is still much to understand about the extent and mechanism of the therapeutic effects of MSCs and stromal vascular fraction cells, and the ideal treatment regimen for each disease type. However, the coming decade will provide long-term follow-up results which will shed light on many of the questions surrounding stem cells and stem cell treatments. Continued research will see MSC therapy become routine and available to all practitioners.

Key Points