Regrow, Repair, Revive: The Rise of Stem Cells in Veterinary Regenerative Medicine

Regenerative medicine is a field of medicine that develops techniques for cultivating, restoring, or replenishing damaged or diseased tissues or organs. Regenerative medicine has gained tremendous pace over recent decades. Stem cells are undifferentiated cells that can renew themselves and transform into tissue cells with specialized functions. Stem cell therapies are thus employed to counteract the body's lack of ability to regenerate damaged tissue and metabolic activity following an acute or chronic injury. The idea of stem cell therapy was first put forward in 1991 by Caplan, who suggested that large-scale differentiation of cells into the target tissue can be obtained by isolation, growth, and expansion of stem cells in in-vitro environments.

Types and Sources of Stem Cells

• Types of stem cells based on their source:

a) Embryonic stem cells (ESCs) 

b) Adult stem cells 

c) Induced Pluripotent Stem Cells (IPSCs)

• Types of stem cells based on the phase of development and differentiation:

        a) Totipotent stem cells

        b) Pluripotent or multipotent stem cells

Totipotent stem cells are found in early embryos at the morula stage before the initiation of gastrulation and can potentially give rise to all embryonic and extra-embryonic tissues. Cell divisions during early embryo development result in the formation of the blastocyst with the pluripotent ESCs found in the inner cell mass. ESC has the potential to generate all body tissue cells except for extra-embryonic tissues and germ cells. With additional development of cells, pluripotent ESCs become progressively less pluripotent and turn into multipotent cells that can differentiate into restricted types of specific cells, commonly based on their germ layer of origin.

The initial isolation of human ESC was reported in 1998. This stimulated many investigations on gene function and expression during embryonic development and cell differentiation processes in addition to the efforts to search for gene targets for novel drugs that could potentially be beneficial for tissue regeneration therapy. The therapeutic broad-spectrum abilities of human ESCs came into conflict with ethical, moral, and cultural issues since their procurement is linked with the killing of human embryos. Thus, other sources of stem cells had to be sought to proceed with the research into stem cell-based therapies. One of the alternatives was created in 2006 by Takahashi and Yamanaka, who converted adult mouse fibroblasts into pluripotent stem cells by retroviral transduction of four particular genes, namely OCT4, c-Myc, SOX2, and KLF4. Such cells were referred to as IPSCs and are analogous to the ESCs in their morphology, growth characteristics, and the expression of ESC marker genes. Another option to ESCs are the stem cells found in adult organisms. Hematopoietic stem cells (HSCs) and non-hematopoietic or mesenchymal stem cells (MSCs) are found in bone marrow and the umbilical cord. These cells are multipotent since they can differentiate into specific body cell types. HSCs can differentiate into various cells of the immune system, erythrocytes, and platelets. MSCs differentiate into bone, cartilage, ligaments, tendons, fat, skin, muscle, and connective tissue cells. MSCs are stimulated endogenously whenever they are required to replace dead, injured, or diseased tissue cells. Studies have revealed that multipotent stem cells can differentiate into osteoblasts, chondroblasts, and adipocytes. In contrast with all other stem cell types, MSCs are identified as the most promising stem cell type to be used in stem cell therapy because of the easy procedures required for their harvesting, isolation, high yield of cells upon harvesting, and the absence of ethical limitations when used. To avert the misapprehension in the arena of adult stem cell research, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) put forth the following set of criteria to define human MSCs both for laboratory-based scientific studies and pre-clinical evaluations.

• MSCs should be plastic-adherent when cultured under standard culture conditions in tissue culture flasks

• 95% of the MSC population should be positive for CD105, CD73, and CD90 and negative for CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA class II

• MSCs should differentiate into osteoblasts, adipocytes, and chondroblasts under standard in-vitro differentiating conditions.

  
  

Clinical Application of MSCs in Veterinary Practice

Stem cells have been employed for the treatment of a wide range of diseases in various animal species, though experimentally. The primary interest of regenerative veterinary medicine was initially addressed to the orthopedic diseases, but currently, the interest is quickly increasing to other conditions like orodental and gastrointestinal tract diseases, liver, kidney, cardiac, respiratory, neuromuscular, dermal, olfactory, and reproductive system diseases. Stem cell therapy has been applied most frequently in dogs and horses for diseases of multiple organ systems and in cats for renal, respiratory, and inflammatory diseases.

Diseases of the Musculoskeletal System

Tendon and Ligament Repair

• The traditional treatments of tendon injury in animals are cooling, bandaging, and rehabilitation with controlled exercise. Pharmacological treatments involve the administration of systemic and local corticosteroids or other anti-inflammatory medications. These conservative methods do not provide full tissue healing, reinjury is frequent, and the animals can't come back to the pre-injury performance level. Optimal treatment must, hence, work towards the regeneration of a normal tendon matrix.

• The use of MSCs has been introduced as an alternative to the traditional approach because it represents a potential tool for better tissue regeneration and aims towards healing with the proper formation of collagen fibers and successful regaining of normal tendon activity with a lesser risk for reoccurrences.

• Tendon-derived stem cells have been described as the best source of stem cells for tendinopathies, but the isolation of stem cells from tendon tissue is extremely difficult. Stem cells from other sources, predominantly from adipose tissue and bone marrow, have been utilized for tendon regeneration.

• Autologous Bone Marrow Mesenchymal Stem Cell (BMMSC) implantation to the horse's superficial digital flexor tendon was first documented in 2003. Following cell injection into 11 racehorses with superficial digital flexor tendon lesions, extensive clinical recovery was noted.

• As with horses, dogs have also undergone experimental MSC therapy. One frequent canine injury is a tear in a cranial crucial ligament of the stifle joint. The rupture thereof is related to stifle osteoarthritis and is responsible for most lameness cases in adult dogs.

  
  

Joint diseases

• Among the most frequent causes of equine athletic career termination and chronic lameness are diseases of joints, the most common being osteoarthritis. Standard treatment of musculoskeletal injury, including damage to articular cartilage, ligaments, and menisci is typically related to poor prognosis for athletic ability of horses.

• The in-vivo efficacy of intra-articular MSC therapy of bone, meniscal, and cartilage disorders in horses has been documented. The best-studied and characterized locomotive system disorder in horses is bone spavin, a degenerative articular disease in which orthodox treatment hinges on the administration of anti-inflammatory corticosteroids for pain and inflammation reduction. Findings from the study in which 16 horses with bone spavin were intra-articularly treated with autologous Adipose Derived Mesenchymal Stem Cells (ADMSCs) indicate the beneficial and long-term effect of MSC therapy. No lameness was noticed 180 days post-treatment in the treated horses compared to the untreated control group. This was verified by scintigraphic study with no evidence of inflammation process in tarsal joints of treated horses.

  
  

Orodental Diseases

• With the increasing development of regenerative cell therapy, stem cells have gained interest in the treatment of orodental tissues. Research emphasizes MSC's immune-modulatory effects to drive regeneration of periodontal and dental tissues and differentiation potential of MSCs for enhancing implant stability and bone tissue repair in alveolar defects.

• Apart from common stem cell sources, including bone marrow and adipose tissue, stem cells derived from local tissues like Dental Pulp Stem Cells (DPSC) or Periodontal Ligament Stem Cells (PDLSC) are investigated as a treatment method in orodental conditions.

• In canine models, Xenogenic Periodontal Ligament MSCs or autologous BMMSCs are advantageous in periodontal ligament reconstruction, in combination with the growth factors, ephrinB2, PRP, or with a porous biphasic calcium phosphate construct.

• Promising outcomes have been reported from the MSC treatment of Feline Chronic Gingivostomatitis (FCGS), a painful and debilitating oral disease of cats, with chronic inflammation of gingiva extending to the buccal and caudal oral mucosa. Arzi et al. demonstrated that IV administration of autologous ADMSCs leads to complete clinical and histological resolution or a decrease in clinical severity of the disease in most cats.

  
  

Digestive tract illnesses

Inflammatory bowel disease (IBD) is a chronic hypersensitivity reaction of autoimmune etiology in the intestinal mucosa. Cats with IBD have been treated with allogeneic ADMSCs, and clinical signs have improved significantly in 5 of 7 cats, as reported by the owners. With their immune-modulatory and anti-inflammatory effects, MSCs would be an appropriate alternative treatment for dogs and cats with IBD. Though preliminary study results are encouraging, more extensive follow-up studies and research are required before MSC treatment can be deemed a safe and effective way to treat IBD in animals.

Liver Diseases

• Yan et al. studied the effect of intravenous injection of autologous ADMSCs in artificially induced acute liver injury in dogs. Peripheral blood liver enzyme levels were decreased, and liver tissue architecture was regained following the therapy, showing a promising application of MSCs in canine liver diseases.

• MSCs were also applied in a liver cirrhosis model in dogs. IV delivery of autologous BMMSCs significantly reduced the extent of the liver fibrosis and enhanced liver function in the cell-treated group with no harmful side effects.

  
  

Renal Diseases

Chronic kidney disease (CKD) is a prevalent geriatric feline medical disorder and is defined by chronic tubulo-interstitial nephritis, interstitial fibrosis, and tubular atrophy. Renal transplantation is, at present, the sole treatment that can potentially reconstitute renal function. Stem cell-based treatments might, thus, offer less invasive treatment alternatives.

In a study by Vidane et al., in cats with spontaneous CKD, allogenic MSCs from the feline amniotic membranes were given intravenously, and after the second intravenous administration of MSCs, there was marked improvement in the renal function.

Cardiac Diseases

• Cardiac stem cell therapies for myocardial repair after a sudden or chronic myocardial infarction in human medicine have been employed for several years.

• Primary myocardial infarction is uncommonly seen in companion animals. Degenerative valvular disease is the most frequent cardiac disease in smaller breeds of dogs and is generally complicated by ventricular dilation and dysfunction. Petchdee and Sompeewong studied the effect of IV injection of stem cells derived from puppy deciduous teeth on the degenerative valvular disease with results of significant improvement in the left ventricular ejection fraction.

  
  

Diseases of the Respiratory System

Barussi et al. investigated the effect of intratracheal administration of bone marrow derived mononuclear cells on the progression of the respiratory inflammation in horses with Recurrent Airway Obstruction (RAO). Comparison between treatment with a single intratracheal administration of autologous cells and oral treatment with dexamethasone indicated that bone marrow-derived mononuclear cells had an improved effect on clinical signs and the inflammatory reaction in horses suffering from RAO. IL-10 levels rose following the cell treatment and were significantly higher than in the dexamethasone-treated control group.

Neuromuscular Injuries and Diseases

• Spinal cord injuries (SCIs) are one of the most prevalent neuromuscular injuries in animals and humans, which frequently lead to permanent disabilities.

• Spinal cord injury in dogs might be caused by trauma or the herniation of a vertebral disc. Autologous BMMSC treatment has been evaluated for spontaneous spinal cord injury following spinal trauma in dogs with locally injected cells via hemi-laminectomy. It has been reported that gait, nociception, and proprioception were moderately improved in treated animals.

  
  

Skin Diseases and Wound Healing

• Failed wound healing usually results from a complex array of poor cellular and molecular responses. It tends to result in a chronic, persistent wound, with pain for the patient. Hence, for treating chronic wounds, MSCs could be a suitable form of treatment because of their regenerative and anti-inflammatory potential.

• Animal model studies have demonstrated the therapeutic effect of MSC treatment in wound healing in goats, sheep, horses, and dogs.

• In addition to wound healing, MSCs have also been used for the treatment of atopic dermatitis, one of the most common skin diseases in dogs. The study included 22 dogs with atopic dermatitis, non-responsive to conventional therapy. Pruritus decreased significantly 1 week after IV administration of allogenic ADMSCs. The remission of clinical signs lasted for at least 6 months, with no adverse side events observed.

  
  

Diseases of the Reproductive System

• Numerous stem cell studies are trying to identify treatments for fertility enhancement, both for commercial use in farm animals and for translation into human medicine.

• Notably, canine sperms appear to be vulnerable to the therapy with ADMSC derived extracellular vesicles (ECVs) during cryopreservation, as supplementation with ECVs suppressed the count of defective sperms in thawed semen.

• In mares, endometriosis is a non-curable degenerative uterine disease and induces significant economic losses in the horse industry. To utilize MSCs' immune-modulation effects in uterine disease, endometrial MSCs were studied and extracted from sows, cows, ewes, goats, and mares. MSCs delivery to the uterus of endometriotic mares has already been suggested, and it has been observed that MSCs are retained within the uterus for up to 21 days following intrauterine administration.

• Besides the pathologies of the reproductive system, MSC therapy is also explored for the possible treatment of mastitis in livestock animals, demonstrating an anti-proliferative effect against mastitis caused by Staphylococcus aureus in cattle and a reparative and anti-fibrotic effect in goat chronic mastitis.

  
  

Conclusions

• Veterinary regenerative medicine is a developing field. There have been remarkable improvements in the development of safe and effective stem cell therapies over the last few years. Encouraging results of MSC therapies have been described, particularly for orthopedic diseases in dogs and horses, but significant improvements in MSC therapy have also been achieved in the treatment of other diseases, including FCGS, IBD, and wound healing. The optimistic results of numerous studies hold high hope for the future of stem cell therapies in treating numerous diseases of animals.

• Although there has been significant progress made in veterinary regenerative medicine during the past few years, the field is still in the beginning stages, and there is much more to be accomplished toward answering many of the questions before effective, standardized therapies can be provided to the clinical patients. We are living in exciting times as emerging regenerative therapies are increasing. One may hope that the ongoing research in this field will bring us to the day when the stem cell therapies for most of today's untreatable illnesses will not be just a probability but a viable and available option for the patients both in veterinary and human medicine.

  
  

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