Biobest Laboratories Ltd, 6 Charles Darwin House, The Edinburgh Technopole, Milton Bridge,Nr Penicuik, EH26 0PY Tel: +44 (0)131 440 2628

Use of Adipose Mesenchymal Stem Cells in Canine Regenerative Medicine

Kieran McDonald BSc (Hons) MSc MIBMS Paul Burr BSc(VetSci)Hons BVM&S PhD MRCVS

Mesenchymal Stem Cells (MSCs) are being increasingly used in clinical applications due to their capacity for self-renewal and multipotent differentiation, as well as their immunomodulatory properties. The willingness of the Veterinary Medicines Directorate (VMD) to permit such novel therapeutic approaches using biologicals with a light regulatory touch, as well as encouraging post treatment data from years of use in the equine sector (1), has allowed the application of these cells to a range of new treatments in companion animals.

There are two approaches for using adipose derived stem cells for therapeutic use. Firstly, the adipose tissue can be collected, processed and returned for implant without culture; this material is referred to as the Stromal Vascular Fraction (SVF). Processing is minimal and can involve a laboratory or can be achieved using an automated system and so be done in-practice. The output of this process will be a pellet of cells ready for implantation but it is worth noting that there will be some variability in the product produced (2). This approach has the advantage that the collection, processing and re-implantation of the cells can be done on the same day, or within 2 days if the sample is sent to a lab for processing, but the disadvantage is that that many of the cells which are then introduced into the site of injury or disease are not stem cells but a heterogeneous population of MSCs along with endothelial cells, smooth muscle cells, fibroblasts, preadipocytes and immune cells (3). An alternative approach is that the cells are isolated and cultured for several days until they number many millions, this highly purified population of cells is then ready to be re-implanted suspended in autologous serum or plasma.

As performed at our laboratory both of these approaches are autologous and so only material removed from the patient at sampling is returned at implant with no extra additives or carrier solutions. Use of SVF allows for a more rapid treatment, and is generally less expensive, but there is a growing consensus that there may be a dose dependant response when using implanted MSCs so it would be reasonable to suggest that the culture of a large number of highly purified cells may prove to be more efficacious in many cases.

There are several sites from which adipose tissue can be collected. Most commonly it is taken from a fat pad located behind the scapula but several sites have been suggested as appropriate sources: retroperitoneal adipose tissue (4), lateral thoracic area (5), gluteous fat (6), and the inguinal region (7). Cells are isolated and (where appropriate) cultured using standard practices (8) and have been shown to

Biobest Laboratories Ltd, 6 Charles Darwin House, The Edinburgh Technopole, Milton Bridge,Nr Penicuik, EH26 0PY Tel: +44 (0)131 440 2628

be present in adipose tissue in large numbers. They have also been observed to show a high proliferative value when compared to those found in bone marrow; an equal volume of adipose tissue can yield 10 million cells in half of the time taken by a bone marrow derived culture. In dogs, adipose derived cells have been shown to be the best available for therapeutic use when compared to bone marrow, Wharton’s Jelly, and umbilical cord blood (9). Furthermore although there is a limited reduction in availability and proliferative potential of MSCs in adipose tissue as animals age, these cells have been shown to be present in therapeutically useful numbers in dogs in their teens.

Indications for use.

A growing number of practices are advertising stem cell treatment in dogs citing joint problems as the main target for the therapy, especially those conditions that end up as arthritis such as hip dysplasia, elbow dysplasia and osteochondrosis. One of the main benefits of this treatment is its utility in intractable cases where pain medication is no longer effective or cannot be used. Repeat applications of cells has been advocated, and this is facilitated by the fact that cells can be stored long term by cryopreservation and so administered to chronic cases without the expense and additional trauma associated with further tissue harvesting and processing.

Secondary to osteoarthritis is the treatment of a range of bone and tendon injuries, and furthermore, on a case by case basis, they have been used in the treatment of a range of other conditions such as inflammatory bowel disease, degenerative myelopathy, acute and chronic renal failure, chronic obstructive pulmonary disease, autoimmune diseases, and spinal trauma. Success of these applications can be difficult to judge but there is some evidence of improvements post treatment, both anecdotal and published (10, 11). A number of studies are currently ongoing to further assess the impact of these cells in regenerative medicine

Current research.

There are a wide range of diseases which are being experimentally treated using stem cells in dogs, many with a view to developing veterinary solutions rather than as animal models. However, only a small number of these studies are in naturally affected dogs, most involved induced lesions (12). There have also been several studies using allogeneic adipose derived stem cells to treat a range of diseases (Keratoconjunctivitis Sicca [13], inflammatory bowel disease [14], hip dysplasia [15] and bone regeneration [16]), however, the use of allogeneic transplants renders it unlikely that this type of approach will be licensed in the UK in the near future as the VMD restricts all procedures using MSCs to autologous cells and has shown no desire to change this.

Regulation

All cell culture must be performed at an Approved Scientific Manufacturing Centre as licensed by the VMD.

Biobest Laboratories Ltd, 6 Charles Darwin House, The Edinburgh Technopole, Milton Bridge,Nr Penicuik, EH26 0PY Tel: +44 (0)131 440 2628

There are a range of possible indications for the use of these cells but as has been the case in the equine sector (where MSCs have been used to treat animals for over 14 years), the Veterinary Surgeon treating the case has overall responsibility for when and how these cells are used. The rapidly increasing scope of the potential application of these cells is pushing the technology on and breaking new ground at a rapid pace.

References

1. Godwin EE, et. al. Implantation of bone marrow-derived mesenchymal stem cells demonstrates improved outcome in horses with overstrain injury of the superficial digital flexor tendon. Equine Vet J. 2012 Jan;44(1):25-32.

2. Aronowitz JA et. al. Adipose stromal vascular fraction isolation: a head-to-head comparison of four commercial cell separation systems. Plast Reconstr Surg. 2013 Dec;132(6):932e-9e

3. Li-Ying Chan L. et. al. Automated Enumeration and Viability Measurement of Canine Stromal Vascular Fraction Cells Using Fluorescence-Based Image Cytometry Method J of Fluorescence. July 2014, Volume 24, Issue 4, pp 983-989.

4. Bigham-Sadegh A. et. al.: Effects of adipose tissue stem cell concurrent with greater omentum on experimental long-bone healing in dog. Connect Tissue Res. 2012, 53:334-342.

5. Haghighat A. et. al.: Adipose derived stem cells for treatment of mandibular bone defects: an autologous study in dogs. Dent Res J (Isfahan) 2011, 8(suppl):51-57.

6. Marx C. et. al. Acupoint Injection of Autologous Stromal Vascular Fraction and Allogeneic Adipose-Derived Stem Cells to Treat Hip Dysplasia in Dogs Stem Cells Int. 2014; 2014: 391274.

7. Guercio A. et. al.: Production of canine mesenchymal stem cells from adipose tissue and their application in dogs with chronic osteoarthritis of the humeroradial joints. Ell Biol Int 2012, 36:189-194.

8. Neupane M. et. al. Isolation and Characterization of Canine Adipose–Derived Mesenchymal Stem Cells. TISSUE ENGINEERING: Part A 2008, Volume 14, Number 6: 1007-1015

9. Kang BJ. et. al.: Comparing the osteogenic potential of canine mesenchymal stem cells derived from adipose tissues, bone marrow, umbilical cord blood, and Wharton’s jelly for treating bone defects. J Vet Sci 2012, 13:299-310.

10. Allan J. et. al. Effects of adipose-derived nucleated cell fractions on tendon repair in horses with collagen-induced tendonitis. American Journal of Veterinary Research. July 2008, Vol. 69, No. 7, Pages 928-937.

11. Black L.L. Effect of adipose-derived mesenchymal stem and regenerative cells on lamness in dogs with chronic osteoarthritis of the coxofemoral joints: a randomised, double blinded, multicenter, controlled trial. Veterinary Theraputics. Vol. 8 No. 4 Winter 2007.

Biobest Laboratories Ltd, 6 Charles Darwin House, The Edinburgh Technopole, Milton Bridge,Nr Penicuik, EH26 0PY Tel: +44 (0)131 440 2628

12. Marx C. et. al. Adipose-derived stem cells in veterinary medicine: characterization and therapeutic applications. Stem Cells Dev. 2015 Apr 1;24(7):803-13

13. Villatoro AJ. et. al. Use of Adipose-Derived Mesenchymal Stem Cells in Keratoconjunctivitis Sicca in a Canine Model. Biomed Res Int. 2015;2015:527926. doi: 10.1155/2015/527926. Epub 2015 Feb 23.

14. Pérez-Merino EM. et. al. Safety and efficacy of allogeneic adipose tissue-derived mesenchymal stem cells for treatment of dogs with inflammatory bowel disease: Endoscopic and histological outcomes. Vet J. 2015 Jul 23. pii: S1090-0233(15)00314-7. doi: 10.1016/j.tvjl.2015.07.023. [Epub ahead of print]

15. Black LL. Effect of adipose-derived mesenchymal stem and regenerative cells on lameness in dogs with chronic osteoarthritis of the coxofemoral joints: a randomized, double-blinded, multicenter, controlled trial. Vet Ther. 2007 Winter;8(4):272-84.

16. Liu G. et. al. Bone regeneration in a canine cranial model using allogeneic adipose derived stem cells and coral scaffold. Biomaterials. 2013 Apr;34(11):2655-64. doi: 10.1016/j.biomaterials.2013.01.004. Epub 2013 Jan 21.

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New customers can obtain one free stem cell culture (equine or canine) by submitting this voucher along with your adipose or bone marrow sample and submission form. If required, sampling kits can be purchased by contacting our office team on 0131 440 2628, submission paperwork can also be

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