1. -Stem Cell Therapy - Introduction and Practical Guide to Use in Small Animal Practice
This is an introductory slide set intended to be used by Vet-Stem credentialed vets in presenting stem cell therapy to other vets.

2. What Do Stem Cells Do?
10 million cells die in your body every minute of every day.
Your own stem cells replace them so you can continue living.
This is what stem cells do for a living.
Simple explanation for what adult stem cells are doing every day in every body.

3. What are Stem Cells?
Stem Cells are Primitive cells present in almost every tissue:
Self Renewing
Able to become different tissue types
Trophic Factories – Growth Factors
These are the three major distinctions of what define stem cells.

4. Embryonic vs Adult Stem Cells
Stem Cells from Embryos
Intended to form whole animal - not for repair
Can form tumors – UNPREDICTABLE
Maybe someday
Adult Stem Cells – many sources
Intended for self repair
Do not form tumors when injected
Now
Gruen L and Grabel L, Stem Cell 2006;24;
Adult stem cells are the bodies natural healing cells for repair. They are found in fat, bone marrow, skin and most other tissues of the body.

5. Enabling Innovation
Dr. Bill Futrell, et al – Univ of Pittsburgh 1998 “Discovered true stem cells in fat that could create new tissues like bone and cartilage”
Stem Cells from fat Extracted can become bone Fix fractures
in the lab
The original discovery of stem cells from fat occurred at the Univ of Pittsburgh in the plastic surgery department by Dr. Bill Futrell and his group. They found that cells derived from extracted fat tissue could become bone, cartilage, fat and nerves. Patents were filed and this field was born. The question was whether we could harness this to fix a clinical problem like a non-union fracture.

6. Why Adipose as Stem Cell Source?
High healing cell count – No culturing required
1000X stem cell concentration as bone marrow
Family of healing cells - heterogeneous
Rapid, Easy to access
Over 1700 peer reviewed papers published

7. What Cells Do We Use?1
Contrary to the older press and literature, approximately 1/3 of the nucleated cells derived from a sample of fat are stem cells. There are also over 28% of the cells that are regenerative progenitor type cells with the ability to provide growth factors and assist in healing. This is over 1000X more concentrated than bone marrow and is the reason you use the cells fresh, without need for cell culture. However, if you run out of fresh or frozen cells, you can grow additional doses from a small retention sample kept on all patients.

8. Mechanisms of Repair Trophic support - growth factors and cytokines
Anti-inflammatory
Differentiation into tissue
Homing to injury site
Immune System Modulation
These are the five main mechanisms of action of adult stem cells. We usually think of differentiation into tissue as the first and most important mechanism, and it is important, but maybe not the most dominant.

9. The Injury Response Cascade
Time
Fibrosis
Inflammation
“Regeneration”
Acute Injury
Scar Complete
Magnitude
QUICK FIX
In a simple schematic, a typical injury causes a regenerative response, for example in a heart attack. The tissue tries to regenerative for a short period of time and then resorts to scar tissue formation to “patch” the injury.
Courtesy, A Caplan, Case Western Reserve

10. Stem Cells in Injury Response
“Regeneration”
Acute Injury
Scar Reduced
Time
Magnitude
Inflammation
Fibrosis
In stem cell therapy, we are attempting to boost the number of stem cells and increase the length of time that the body attempts to regenerate a more normal/natural tissue in its repair.
Stem Cells
Courtesy, A Caplan, Case Western Reserve

11. Roles / Functions
“Stem cells are injury-specific, perfectly choreographed pharmaceutical factories”
Dr. Arnold Caplan
Case Western Reserve
Influence by injury micro-environment
Stem cells respond to specific injury micro-environment.

12. Stem Cells and Joint Therapy Mechanisms
Chondrogenesis1 - new cartilage formation
Lubricin2 - lubricate joint, improve range of motion
IRAP Secretion3 - blocks IL-1 inflammation mediator
Decrease Inflammation4 - reduce pain and swelling
So how do we believe these cells are working in the joints. First, arthroscopy data shows that new cartilage can and is formed. But many times the therapy seems to work much faster than one would expect from this mechanism. Lee et all showed in 2008 that stem cells can produce Lubricin, a key joint lubricating molecule. Ortiz et al in 2007 published that stem cells can also produce IRAP, a potent molecule that blocks IL-1 action, a major contributor to joint inflammation and cartilage degradation. Finally, Tholpady and many others have shown that stem cells can produce a range of anti-inflammatory cytokines and down regulate inflammation.
1 Wei et al, Cytotherapy, 2007
2 Lee et al, BBRC, 2008
3 Ortiz et al, PNAS 2007
4 Tholpady, Plast Surg, 2006

13. Evidence-based Cell Therapy
DATA TYPE AVAILABLE
Canine Osteoarthritis
Canine Tendon and Ligament
In-vitro (lab bench) X
Lab Animal
Case Studies
Retrospective Studies
Non-Random, Prospective
Randomized, Controlled
(equine)
Human Clinical Studies
STATUS
Supported
Today we talk about evidence-based practice of medicine. So what types of evidence do we have to support this type of adipose stem cell therapy. For canine arthritis we have in-vitro, lab animal, case studies, retrospective studies, non-randomized but prospective studies, randomized placebo controlled blinded studies, and human clinical studies. For soft tissue tendon and ligament injury, we have the same data, but the controlled, randomized studies we have are equine only.

14. Rabbit Osteochondral Defect Repair Model
Nathan et al, “Cell-based therapy in the repair of osteochondral defects: A novel use for adipose tissue” Tissue Engineering, 2003.

15. Elbow Dysplasia - DJD Annie – 9 YO
Chronic elbow dysplasia – non-NSAID controlled pain
Stem Cell Therapy at Day 0 and 14.
Now able to jump in and out of car; took 2 hour run in the woods before her 90 day study exam!

16. Bilateral Stifle / Hips
4 YO Rat Terrier
Bilateral stifle and hip degeneration since 6 months old
NSAIDS for 12 months and still painful
===============
Intra-articular RX with Stem cells in all four joints

17. Bilateral Stifle / Hips
4 YO Rat Terrier
Post Treatment:
2 weeks – Dramatic pain reduction
Now NSAID free for >24 months

18. Human Therapy
“Autologous stem cells (adipose) and fibrin glue used to treat widespread traumatic calvarial defects: case report”
A young girl with major trauma to skull cap that could not be repaired with bone graft was treated with her own adipose stem cells and re-grew the skull bones.
Journal of Cranio-Maxillofacial Surgery (2004) 32, 370–373
One of the first reports confirming this creation of tissue in a human came in A young girl had lost most of her normal skull cap due to a major trauma and traditional bone grafting had failed. With a single application of fresh, adipose-derived stem and regenerative cell mix, they were able to grow back the majority of her calvarium.

19. 180 Day Prospective, Non-randomized Study Uni or Bilateral Elbow OA
Intra-articular stem cells 1x
Vet and Owner assessments at pre, 30, 60, 90, 180
14 dog study
The first study to review is a prospective study, but without controls, using baseline values as comparison. 14 dogs were treated in 5 referral clinics in the US. A single intraarticular injection was given and dogs were followed for 180 days.

20. All measured outcomes were improved from baseline.

21. 180 Day Study – Blinded/Placebo Bilateral Hip OA – 18 dogs
Intra-articular stem cells – 1X
Vet/Owner assessments at pre, 30, 60, 90
The second OA study to review was published in Veterinary Therapeutics in 2007 and is a double-blinded, randomized, placebo-controlled study of OA in the hip. 18 dogs were treated for bilateral hip OA with a single intraarticular injection per joint. The study was blinded for 90 days.

22. This graph shows the improvement over the 90 day period in treated versus saline-injected control dogs. You can see there was a placebo effect, highlighting why placebo-controlled studies are important. The stem cell treated group out-performed the saline group at all time points.

23. Chronic Knee – Post-surgical OA
Pilot Study (n=9)
180 Day Pilot Study, 9 dogs
Chronic Post-Surgical OA (>3 Mo)
Intraarticular stem cells – 1X
Vet and Owner assessments at pre, 30, 90, 180
Not Blinded
The last OA study in dogs to review was done in a single clinic by a single surgeon. All dogs had previous surgery in the stifle, had not responded favorable, and now had OA. They had all been re-scoped to confirm that no additional surgery would help. A single injection was made into each joint and improvements from baseline over 180 days were made.

24. Again, all parameters measured improved
Again, all parameters measured improved. “V” means the veterinarian measured the lameness, range of motion and pain and “O” indicates owner measured.

25. Tendon / Ligament Clinical Data
Equine – Blinded, placebo-controlled study1
Equine – Retrospective2 – tendonitis
Equine – Retrospective3 - ligament therapy
Canine – Clinical case studies4
Canine – Retrospective analysis5
Canine – Prospective, non-randomized6 – shoulder instability
In the area of tendon/ligament repair, there are equine and canine data that demonstrate repair of the joint-related soft tissue by adipose stem and regenerative cells.
1 Nixon et al, AJVR, 2007
Meredith et al, ACVIM, 2006
Harman, VOS, 2007
Bausman, IFATS, 2008
5 Harman et al, IFATS, 2007
6 Canapp, AVMA, 2009

26. 8 Horses – 4 treated / 4 controls
Collagenase induced injury – Rx 10 days later
Fully blinded histology
Controls were treated with same volume of saline
This is the first reported blinded, placebo controlled study of adipose stem cells in tendon repair in the horse. It was conducted at Cornell University by Dr. Linda Dahlgren and staff in Dr. Alan Nixon’s lab. 8 horses were treated in two groups. Controls were blinded and saline injected. The injury was created by injection of collagenase into the tendons of lab horses. The injury was not treated with stem cells until 10 days after the injection.

27. Outcome: Less scar - More normal tendon
Stem Cell Treated
Saline Control
Outcome: Less scar - More normal tendon
Statistically improved tendon healing

28. Why Use Vet-Stem? Began treating horses in 2003
Global patents/licenses (over 55 issued patents)
Over 3,000 horses and 2,000 dogs treated
Global leader in stem cell therapy
Peer-reviewed, published controlled studies
High quality laboratory
Human stem cell alliances

29. Safety Profile* Horses: 7/3,359 (0.2%) Only local swelling/pain
No systemic effects
Dog: /1,695 (0.1%) Only local swelling/pain
*probable adverse events, through 6/1/09
Always we should ask about the risk/benefit profile for any therapy. Since the cells are from the same animal, rejection and disease transmission are minimized. In horses, 7/3,359 treated horses have had a local reaction that could be probably attributed to the cell therapy injections. No systemic reactions have been seen. In the dog, 2/1,695 dogs treated showed a local joint reaction after treatment that could be probably attributed to the cell injections. Overall, a very nice safety profile.

30. Quality Standards Written standard operating procedures
Independent Quality Assurance Unit
Routine audits of all procedures
Sample release criteria
Formal training systems
Environmental monitoring
All Handling Done In Sterile Hoods
Vet-Stem has strict quality standards and we follow the FDA Good Tissue Practices Act as it applies to autologous cell therapy.

31. Resources from Vet-Stem
Technical training – course & consultation
Case selection - consultation
Cell banking
Lifetime cell supply – Cell Culture Service
Marketing Kit
Owner support
Clinic DVD – waiting room
Brochures, posters, counter-top displays
Power Point presentations
Co-op Advertising
Newsletters

32. Keys to Success Credentialing Course (www.vet-stem.com)
Complete Diagnostic Work-up
Risk Assessment of Concurrent Diseases
Proper Collection – close dead space
Proper use of Kit
Proper Injection Technique
Follow up and Rehab Program

33. Case Selection for OA Ideal Stem Cell Case
Clearly defined disease – lameness
Non-surgical candidate – surgery first, if needed
Limited intra-articular osteophytes
One or two joints
No other major systemic diseases
No major spinal disease
NSAID – non-responsive or lack of tolerance
Owner has rational expectations
For osteoarthritis, these are the best criteria for selection of the ideal case.

34. Cruciate Ligament Diseases and the Uses of Stem Cells
Surgery – always assess need and make appropriate surgical repairs
At surgery – adjunct to reduce inflammation and scar tissue
After surgery – treat the synovitis and encourage cartilage repair
Chronic ACL inflammation and synovitis
Cruciate repair in the dog should be approached with caution. If the case needs surgery, stem cells are not a replacement. If the injury is less than a 25% repair, then stem cells may be indicated to stimulate repair of the torn cruciate. If the joint injury is older and already has OA, then stem cells can be used to treat the pain, inflammation and damage. Stem cells are a great adjunct therapy to surgical repair as well. 34

35. Current Supported Indications Canine/Feline
Osteoarthritis
Polyarthritis
Tendonitis
Ligament injury

36. Service Overview
The Vet-Stem Service is simple. The credentialed veterinarian collects a small sample of fat (60-90 grams), sends it by Fed Ex to the Vet-Stem lab in San Diego. The cell technicians will receive the fat, extract the cells, do quality checks and counts, and send cells back THE SAME DAY with the appropriate number of doses loaded into syringes in the appropriate volume for each patient. The Vet will receive the return FedEx shipment approximately 48 hours after collection, and inject the cells into the injured joint, tendon, or IV. Any excess cells are banked at Vet-Stem and stored in liquid nitrogen ready for use in the future.

37. Vet-Stem Adipose Processing Lab
1. Immediate use
Summarizing, the lab sends back doses ready to inject for immediate use and banks any extra cells for future use. The number of cells for any individual animal depends on the amount of fat submitted and on the number of cells/gram of fat for that individual animal. Animals with systemic disease such as Cushing's tend to have a lower number of cells/gram of fat.
3. Lifetime supply by culture
2. Frozen for future

38. Real World Dog OA Cases
This graph shows the improvement in the first 90 days after treatment of a large group of commercial dogs. The data was retrieved by survey from the attending vets. You can see that the dogs went, on average, from a moderate degree of pain, lameness and range of motion to between normal and mild.

39. Change in NSAID Usage in Dogs Treated with Stem Cells for Osteoarthritis
90 Days Post Stem Cell Treatment
246 Days Post Stem Cell Treatment
survey range: days, N= 170
owner voluntary responses 10/08- 05/09
survey range: days, N= 139

40. Conclusion: At 90 and 246 days after treatment
Greater than 33% of dogs discontinued the use of NSAIDs completely
Greater than 28% of dogs decreased their dependency on NSAIDs

41. This graphic demonstrates that over 80% of dogs showed an improvement in quality of life according to survey results from owners AND vets. Additionally, over 85% of owners would recommend Vet-Stem therapy to others.

42. Media Coverage of Veterinary Medicine

43. Additional Slides
The additional slides that follow are intended to be supplementary information and can be used to emphasize a particular area of the talk or to modify the theme or direction of the talk for the intended audience.

44. Mechanisms of Repair 1. Trophic support – growth factors and cytokines
Anti-scarring - MMPs (reduce scar tissue formation)
Angiogenic - VEGF (induces new blood vessels)
Anti-apoptotic (block cell death after injury)
Stimulation of resident tissue stem cells
Caplan and Dennis, J Cell Biochem, 2006
Anti-scarring – these cells produce MMP, matrix metaleoproteinases, that are natural scar tissue remodelers. The cells produce VEGF, vascular endothelial growth factor, that induces growth of new blood vessels. Anti-apoptosis – this is programmed cell death from injury like ischemia – stem cells via growth factors and cell-to-cell transfer of mitochondria can “rescue” dying cells. They also stimulate stem cells resident in the injured tissue to begin dividing and assisting in the healing/regeneration.

45. Mechanisms of Repair 2. Anti-inflammatory
Decrease pro-inflammatory mediators
Increase anti-inflammatory mediators
Tholpady et al, Clin Plastic Surg, 2006
Stem cells truly down regulate and buffer an over-active inflammatory reaction. Decrease of TNF alpha and interferon gamma are examples of turning down the proinflammatory cytokines.

46. Mechanisms of Repair 3. Differentiation into tissue Nerve Bone
Cartilage
Liver
Fat-derived
Stem Cells
Cardiac
Fat
Angiogensis/
Anti-apoptosis
Gene Therapy
Muscle
Clearly, in the lab, we can create all these tissues from adipose-derived or bone marrow derived stem cells This has been proven many times in many labs around the world.
Tholpady et al, Clin Plastic Surg, 2006
(Photo courtesy Cytori Therapeutics)

47. Mechanisms of Repair 4. Homing to injury site Damaged cartilage MSCs
This slide demonstrates that MSCs, or mesenchymal stem cells, are able to home to a damaged site in cartilage, even down into the fibrilated cartilage.
Photo Courtesy Cognate Therapeutics

48. Mechanisms of Repair 5. Immune System Modulation
Stem cells can dramatically modulate immune system function. In this example reported in 2009, non-expanded adipose stem cells were used to treat Multiple Sclerosis patients with just an intravenous treatment which dramatically reduced the symptoms, pain, and dysfunction caused by MS autoimmune attack on nerve sheath myelin.
Stem cells down-regulate the immune system attack on its own nerve sheath myelin.

49. Composite Score: Lameness at walk Lameness at trot
Five parameters were measured by the veterinarian and this graph shows the composite scores and the significant differences between treated and placebo-controls
Composite Score: Lameness at walk
Lameness at trot
Pain on manipulation
Range of motion
Functional disability

50. Vet-Stem Adipose Collection Kit
This is the simple collection kit with an ice block (freeze 24 hours before use), sterile collection tubes, and your paperwork for lab submission. Call to schedule the case so the lab is ready for receiving your sample.
Sterile Collection tubes
Cryo-block
Submission form
Owner Consent form

51. Adipose Tissue Harvest Falciform Fat
Most preferred
collection location due to low risk of seroma and adequate fat source
Fat can be collected from falciform ligament. This reduces the chance of a dead space seroma formation and generally yields the highest volume of fat, even in a think dog.

52. Adipose Tissue Harvest Thoracic Approach
As an alternative, the lateral thorax, behind the shoulder, high on the chest wall, is a good site.