Stem cells have the capacity to self-renew to produce identical copies of themselves and to turn into other cell types. They are therefore considered to have great therapeutic potential in helping tissue repair or regeneration. Stem cells from embryos are pluripotent, which means they can turn into any cell type in the body. Adult stem cells, found in small numbers in most, if not all, tissues, are multipotent, meaning they give rise to a range of cell types, depending on their origin. Those found in bone marrow stroma, umbilical cord blood and adipose tissue are mesenchymal stem cells. These turn mainly into connective tissues such as bone, cartilage and tendon for tissue renewal or repair. In veterinary medicine, mesenchymal stem cells from adipose tissue are the most common source for ‘stem cell’ therapy.
In dogs, a sample of adipose tissue is removed under general anaesthesia. The sample is sent to a specialist company for isolation and expansion (multiplication) of ‘stem cells’. The mononuclear cell population is first isolated from the other tissue components (e.g. by enzymatic digestion of solid tissues). Further processing separates out the stem cells; the cells that adhere to plastic used in tissue culture and that undergo self-renewal are selected as the ‘stem cell population’. The degree of purity of stem cells in these cultures is unknown and research continues to try to find markers that can be used to specifically identify mesenchymal stem cells (Guest et al. 2008; Pascucci et al. 2011; Radcliffe et al. 2010) .
The isolated ‘stem cells’ are than incubated to allow multiplication. It takes around 2 weeks for the number of cells to multiply to reach the numbers usually used clinically (typically in the order of 2–10 million, although the optimal number is unknown). The purified and expanded ‘stem cell’ population is then returned to the vet for implantation in the patient. Added ingredients, shipment conditions, storage, implantation technique, and factors in the animal’s treatment (such as concurrent use of local anaesthetics) can affect stem cell viability and function (Broeckx et al. 2013; Garvican et al. 2014) . The delay between sampling and treatment prevents the immediate treatment of acute injuries. However, purified populations of stem cells from an animal can be stored in liquid nitrogen for future use to allow the immediate treatment of injury in that animal. The use of an animal’s own cells in this way is called autologous adipose-derived mesenchymal stem cell therapy. Several companies in the UK offer this service for cats and dogs. They include: Biobest Laboratories, Cell Therapy Sciences and Veterinary Tissue Bank.
Products are being developed in an attempt to standardise, and gain marketing authorisation for, ‘stem cell’ therapy (Harman et al 2016). This involves acquiring, purifying and expanding stem cells from a single purpose-bred donor animal. These are allogeneic adipose-derived mesenchymal stem cells. This avoids the need to remove adipose tissue from patients and enables acute treatments. Clearly, there is a need to avoid transmission of infectious agents through such products. No such licensed product is yet available in the UK.
Some trials have used a mixture of cells isolated from adipose tissue, the so-called stromal vascular fraction (Black et al 2007). This heterogenous mixture of cells includes fibroblasts, pericytes, endothelial cells and blood cells as well as mesenchymal stem cells (which have not been cultured and expanded).
Recently a technique called Lipogems has become available for the same-day treatment of dogs (Vet Times 2017). The procedure involves collection of adipose tissue under general anaesthesia, processing of the tissue using a device in the veterinary practice and implantation, possibly while the dog remains under anaesthesia. The procedure is said by the company to work on the principle that mesenchymal stem cells in the body originate as pericytes (cells which line the walls of microvessels and capillaries); the technique is said to not break down the adipose tissue into single cells, but leaves the pericytes in contact with the vessels. The resulting product is re-injected at the site of injury where it is believed that the pericytes become activated to produce mesenchymal stem cells and aid tissue regeneration (Tremolada et al. 2016).
The mechanism of action of stem cell therapy is not completely understood. Originally it was believed that the implanted cells turned into replacement tissue (such as cartilage or bone). Now it is thought that ‘stem cells’ (implanted or endogenous) secrete substances such as cytokines and growth factors that suppress the action of inflammatory cells and promote tissue repair (Black et al. 2007; Al Delfi et al. 2016) . This finding has been reported for mesenchymal ‘stem cells’ in various species and as a result the scientist who originally discovered these cells has called for their name to be changed to “medicinal signalling cells” (Caplan 2017).
Administered ‘stem cells’ fulfil the definition of a veterinary medicinal product and so need a marketing authorisation. This means evidence of safety and efficacy is needed in order to market them in the UK. Currently no stem cell product has a marketing authorisation in the UK. The use of such therapy is permitted under the prescribing cascade as an extemporaneous preparation if there is no suitable veterinary medicine authorised in the UK to treat the condition (VMD 2015).
Currently, any unit processing autologous expanded equine stem cells for clinical use must be authorised by the Veterinary Medicines Directorate (VMD) as an Equine Stem Cell Centre. Authorisation is granted if the VMD is satisfied about the welfare of the animals, consistent and safe production processes, and supervision; it does not guarantee standardisation of cell populations or proof of efficacy. Dog or cat stem cells are not covered by this authorisation scheme and companies offering processed stem cells for clinical use in cats and dogs do not need authorisation by the VMD.
Lipogems is considered to be a medical device not a veterinary medicine (VMD - personal communication) and there is no specific legislation for devices intended for veterinary use. The product carries a ‘CE’ (Conformité Européenne) mark, which indicates that the product meets the Essential Requirements in the relevant European Directive for that device: is safe and fit for its intended purpose; and comes with appropriate labelling and instructions for safe use (Gov.UK 2012; DTB 2010). There is no requirement for evidence of efficacy for a device to be marketed.
Allogeneic adipose-derived stem cell therapy
A blinded randomised placebo-controlled trial recruited client-owned dogs with osteoarthritis in one or two joints and persistent pain and/or lameness for at least 3 months. 74 dogs received an intra-articular dose in one or two joints of 0.7mL saline or mesenchymal stem cells (containing a target dose of 12 million viable stem cells in 0.7mL of a solution containing 10% dimethylsulfoxide preservative) (Harman et al. 2016). The primary outcome measure was owner-assessed change in three predefined activities judged to be affected by osteoarthritis with treatment success defined as a decrease of at least 2 points in the total score (a maximum of 12 points)* at day 60. More of the dogs that received stem cells were judged treatment successes (79% vs. 55%; p=0.03). The absolute difference (24%) translates into a number needed to treat of 5. This means 5 dogs would need to be treated with this stem cell preparation for one dog to achieve at least a 2-point reduction in the overall client-specific outcome measure score after 2 months. There were also improvements in vet-assessed pain on manipulation (93% vs. 50%, p= 0.02) and veterinary global score (87% vs. 31%, p=0.01) but the difference in owner-assessed global score (77% vs. 59%) was not significant. There was a large caregiver placebo effect, which is typical in osteoarthritis therapy, and the possibility that intra-articular dimethylsulfoxide might have had an anti-inflammatory effect cannot be ruled out.
*the client-specific outcome measures scoring system for each of the three activities was as follows: 0 – no problem; 1 – mildly problematic; 2 – moderately problematic; 3 – severely problematic; 4 – impossible.
Autologous adipose-derived stem cell therapy
A blinded randomised study in 39 dogs with hip osteoarthritis compared the effect of a single intra-articular injection of mesenchymal stem cells to a single injection of autologous plasma rich in growth factors (Cuervo et al. 2014) . Significant improvements were reported following both treatments after 1, 3 and 6 months based on the following outcome measures: functional limitation, range of motion, and owner and vet visual analogue scale for pain and quality of life. However, the trial had several methodological problems: growth factor-rich plasma is not a standard or proven therapy, there were several primary outcome measures (increasing the possibility that a significant difference will occur due to chance), and no sample size or power calculations.
In a study in 10 dogs with severe bilateral knee osteoarthritis, 2 to 3 million mesenchymal stem cells were injected into one knee, while the second knee in each dog was injected with sterile phosphate buffer which served as a placebo control (Mohoric et al 2016). One year later, limping (as assessed on the Brunneberg scale) was improved in all but one dog; on radiograph evaluation all placebo-treated joints had worsened while 7 out of 10 stem cell-treated joints showed no worsening (p<0.001). There are several limitations with this study, including that it might not be possible to distinguish between the effects on limping of treatment and placebo.
In a non-randomised trial, 10 dogs with osteoarthritis associated with bilateral hip dysplasia received an intra-articular implantation of 15 million mesenchymal stem cells. (Vilar et al 2016) They were assessed over 6 months subjectively (with visual analogue pain scores and a combination of radiograph and limb function) and objectively (with force plate analysis). The results were compared with those from 5 sound dogs. The treated dogs showed an improvement on subjective measures from 1 month after treatment which continued to 6 months. However, on objective measures the improvement seen in the first month after treatment had disappeared by 6 months.
In a blinded randomised controlled trial 21 dogs with bilateral hip osteoarthritis received an intra-articular injection of mixed adipose-derived cells, or placebo, in each hip (Black et al. 2007) . Compared to placebo there was a statistically significant improvement in investigators’ scores for lameness at trot (at 30, 60 and 90 days after treatment), range of motion (at 30 and 60 days), and pain on manipulation (at 30 days only). However, according to owners’ judgement, there was no significant change in scores between treated and control dogs.
‘Stem cells’ have been used to treat a variety of conditions in dogs, including tendon and ligament injuries (Muir et al. 2016; Canapp et al. 2016) , dry eye (Bittencourt et al. 2016) , spinal cord disease (Besalti et al. 2016; Kim et al. 2016), kidney disease (Lim et al. 2016) and inflammatory bowel disease (Perez-Merino et al. 2015), but these were not randomised controlled trials and so it is not possible to draw conclusions about efficacy.
We could find no published clinical trial evidence on the efficacy of minimally processed adipose tissue (Lipogems) in dogs.
There are a few small published randomised controlled trials investigating the effect of allogeneic mesenchymal stem cells in the treatment of kidney disease in cats, but they did not show beneficial effects (Rosselli et al. 2016; Quimby et al. 2015).
‘Stem cell’ treatments involve the inherent risks of intra-articular injection and (for autologous treatments) anaesthesia and surgery to remove adipose tissue. There is also the potential for stem cells to cause tumour formation, to differentiate into inappropriate cell types and to migrate inappropriately, and for allogeneic cells to stimulate immune responses. Assessment of the adverse effects with long-term follow up is therefore important. So far, experience with ‘stem cells’ has not indicated any worrying safety issues.
In dogs, in the 60-day placebo-controlled trial of allogeneic stem cells there were no significant differences in reports of adverse events between the treatment and control dogs and no serious events deemed to be related to, or caused by, the stem cell injections (Harman et al. 2016).
In 141 racehorses treated with autologous bone marrow-derived mesenchymal stem cells and followed for at least 2 years in an uncontrolled trial, there was no evidence of inappropriate tissue or tumour formation (Godwin et al. 2012) .
In humans, the safety of autologous bone marrow-derived mesenchymal stem cells used intra-articularly for cartilage repair or osteoarthritis treatment was assessed in a systematic review. The review included 8 trials involving a total of 844 procedures with a mean follow-up of 21 months. Four serious adverse events were reported: one infection following bone marrow aspiration which resolved with antibiotics; one pulmonary embolism possibly related to bone marrow aspiration; and two tumours, not at the site of injection, that were considered unrelated to treatment. Twenty-two other cases of possible procedure-related adverse events were documented, plus seven possible stem cell-product related events. Increased pain and swelling was the only adverse event reported as related to the stem cell product (Peeters et al 2013).
In the UK, mesenchymal ‘stem cell’ therapy is available for cats and dogs via specialist companies that can process an adipose tissue sample to culture and multiply mesenchymal stem cells for implantation in the patient (autologous or self therapy). ‘Stem cell’ therapy is considered a medical treatment but there are no licensed forms available in the UK. How this therapy might work is not fully understood and it now appears that administered mesenchymal cells might secrete anti-inflammatory and growth factors rather than differentiating into tissue cells, leading some to suggest that the cells should be named medicinal signalling cells rather than mesenchymal stem cells.
Randomised controlled trials in dogs of single doses of autologous ‘stem cell’ therapy in the treatment of osteoarthritis have not shown clear benefits. One fairly large placebo-controlled study using donor (allogeneic) ‘stem cells’ found that the treatment significantly improved owner-assessed activities, albeit with a large placebo effect; the possibility that a preservative included with the stem cell preparation might have contributed to the efficacy cannot be ruled out. How the treatments compare with NSAIDs or joint replacement surgery is not known.
There is no convincing evidence of the efficacy of ‘stem cell’ therapy in the treatment of other conditions in dogs or in cats. There is no evidence of the efficacy of minimally-processed adipose tissue (Lipogems) in the treatment of dogs.
There has been little evaluation of the safety of ‘stem cell’ therapy, including in the long term, although experience so far in dogs, horses and humans does not suggest safety problems.
Owners who are interested in ‘stem cell’ therapy for their pets should be told about the uncertainties about its effectiveness and safety. Ideally ‘stem cell’ therapy should only be used as part of blinded randomised controlled trials in order to develop knowledge about this form of treatment.
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