1. The Concepts Vet-Stem Credentialing Course
12/13/12
The Concepts
Vet-Stem Credentialing Course
Veterinary Regenerative Medicine 101
Veterinary Regenerative Medicine 101

2. Regenerative Medicine
12/13/12
What should you ask about any new therapy?
Evidence of potential mechanisms
Evidence of efficacy
Evidence of safety
Formulary for how to use in practice
Veterinary Regenerative Medicine 101

3. Course Outline VRM 101 The Concepts
12/13/12
VRM 101 The Concepts
VRM 102 Evidence-Based Regenerative Medicine – Pain and Orthopedic Use
VRM 103 Stem Cell Mediated Regeneration
VRM 104A Small Animal Clinician’s Approach
VRM 104B Equine Clinician’s Approach
VRM 105A Small Animal Adipose Harvest / Injection
VRM 105B Equine Adipose Harvest / Injection
Veterinary Regenerative Medicine 101

4. Regenerative Medicine
12/13/12
Module Outline
Discover the meaning of “Regenerative Medicine”
Stem Cells – What really are they?
Learn the sources and types of stem cells
Explore the mechanisms of action of stem cells
Veterinary Regenerative Medicine 101

5. What is Regenerative Medicine?
12/13/12
10 million cells die in your body every minute of every day.
Your own stem cells replace them so you can continue living.
Goal of Regenerative Medicine:
Restitutio In Integrum
Restore to Original
Simple explanation for what adult stem cells are doing every day in every body.
Veterinary Regenerative Medicine 101

6. Regenerative Medicine
12/13/12
Damage
Repair
Regenerate
An analogy. Damage to a roof. Then repair, or patch of the damage. But regenerate is to return to normal architecture.
Veterinary Regenerative Medicine 101

7. Why Use Regenerative Medicine?
Current research and clinical trials are exploring regenerative medicine for nearly every organ system. Examples are:
Osteoarthritis
Tendon/ligament injury
Renal failure
Liver failure
Laminitis
Immune-mediated diseases: atopy, IBD, COPD

8. Pericytes on blood vessels. Courtesy Arnold Caplan and Bruno Peault
What are Stem Cells?
12/13/12
Stem Cells are:
Primitive cells present in almost every tissue
Pericytes on blood vessels. Courtesy Arnold Caplan and Bruno Peault
Veterinary Regenerative Medicine 101

9. What are Stem Cells? Stem Cells are: Tendon, Ligament, Bone
12/13/12
Stem Cells are:
Primitive cells present in almost every tissue
Able to become different types of tissue:
Tendon, Ligament, Bone
Stem Cells are able to become different types of tissue: tendon, ligament, bone. This picture is a dish of growing stem cells with growth factors to induce them to become cardiomyocytes.
Stem cells differentiated into cardiomyocytes using growth factors. Courtesy NIH.
Veterinary Regenerative Medicine 101

10. What are Stem Cells? Stem Cells are: Tendon, Ligament, Bone
12/13/12
Stem Cells are:
Primitive cells present in almost every tissue
Able to become different types of tissue:
Tendon, Ligament, Bone
Stem Cells are able to become different types of tissue: tendon, ligament, bone. This picture is a dish of growing stem cells with growth factors to induce them to become cardiomyocytes.
Stem cells differentiated into cardiomyocytes using growth factors. Courtesy NIH.
Veterinary Regenerative Medicine 101

11. Dividing stem cells. Courtesy Salk Institute.
What are Stem Cells?
12/13/12
Stem Cells are:
Primitive cells present in almost every tissue
Able to become different types of tissue:
Tendon, Ligament, Bone
Self-renewing
The third characteristic of stem cells is that they can self renew. This video showing dividing neural stem cells.
Dividing stem cells. Courtesy Salk Institute.
Veterinary Regenerative Medicine 101

12. What are Stem Cells? Stem Cells are: Tendon, Ligament, Bone
12/13/12
Stem Cells are:
Primitive cells present in almost every tissue
Able to become different types of tissue:
Tendon, Ligament, Bone
Self-renewing
Pharmaceutical Factories
The fourth and most surprising characteristic of stem cells is that they are virtual trophic factories producing many growth factors and cytokines in response to injuries or disease.
Veterinary Regenerative Medicine 101

13. Definitions Multipotent turn into any cell line of same germ layer
Pluripotent turn into any cell line except placental
Totipotent turn into any cell type including placental
Autograft from animal A, into animal A
Allograft from animal B, into animal A
Xenograft from species B, into species A
Mesenchymal originating from mesoderm

14. Mesenchymal stem cells
A Rose By Any Other Name
Stem cells aka…
Mesenchymal stem cells
Mesenchymal Stromal cells
Multipotent / Pluripotent cells
Stromal vascular fraction
Nucleated fraction

15. Embryonic vs Adult Stem Cells
Source: early embryo
ethical dilemma
Differentiate into all tissues
Purpose: form organism
Form Teratomas UNPREDICTABLE
Adult
Source: all adult tissues (?)
no ethical dilemma
Differentiate into most tissues
Purpose: Regenerate
No evidence of Teratoma formation
Emphasize V et-Stem doing ONLY Adult
Gruen L and Grabel L, Concise Review: “Scientific and Ethical Roadblocks to Human Embryonic Stem Cell Therapy.” Stem Cell 2006;24;

16. How to Use Stem Cells Cell Therapy NOW Tissue Engineering
12/13/12
Cell Therapy
Injection of non-differentiated cells
Cells coordinate healing and regeneration
NOW
Tissue Engineering
Growing tissues and/or organs ex-vivo
Stem cells differentiated on a scaffold then implanted
Potential Future
Cell therapy is NOW. Tissue Engineering, for all the $$$ spent, still has not really delivered many success stories.
Caplan, J Cell. Physiol. 2007, 213:
Veterinary Regenerative Medicine 101

17. Stem Cell Mediated Regeneration
12/13/12
Homing (like WBC)
Differentiation
Direct differentiation into needed cell types
Recruit and stimulate mitosis of local progenitor cells
Trophic support - growth factors and cytokines
Block pain (opioid receptor agonist)
Down-regulate inflammatory mediators
Block cell death (anti-apoptosis)
Stimulate angiogenesis
Anti-fibrosis (block scar)
Veterinary Regenerative Medicine 101

18. Homing Mechanism – Fracture Model
12/13/12
Homing of luminescent adipose stem cells to fracture site from IV administration.
S-W Lee et al, J Ortho Res, 2009
(Stanford Univ)
A fracture is created in the femur of treatment rats and no fracture in the controls. The cells clearly home to the fracture.
Veterinary Regenerative Medicine 101

19. Cruciate Ligament - Chondroprotection
12/13/12
Sham Surgery ASC IA Control
Above toluidine blue staining of cartilage surface at 20 weeks after cranial cruciate ligament transection. Only treatment was group B given 1MM adipose-stem cells by intraarticular injection with no carrier/scaffold.
This study is closer to canine OA. The rabbits were given a cruciate ligament transection and then either saline control or 1M ASC intraarticular. It is clear to see that the ASC group had substantially protected cartilage surfaces and that the controls had very severe damage to the surfaces. This speaks to the possibiilty of chondroprotection and disease-modifying effect of stem cells.
Toghraie et al, “Treatment of osteoarthritis with infrapatellar fat pad derived mesenchymal stem cells in rabbit” The Knee 2011;1:71-75.
Veterinary Regenerative Medicine 101

20. Pain Relief Mechanisms
12/13/12
Eaton M. Cell and molecular approaches to the attenuation of pain after spinal cord injury. J Neurotrauma 2006;23(23/4):
Guo – Bone marrow stromal cells produce long-term pain relief in rat models of persistent pain. Stem Cells 2011;29(8):
Klass M, Gavrikov V, Csete M et al. Intravenous mononuclear marrow cells reverse neuropathic pain from experimental mononeuropathy. Anesth Analg 2007;104:
Malik RA, Veves A, Tesfaye S. Ameliorating human diabetic neuropathy: Lessons from implanting hematopoietic mononuclear cells. Exper Neuro 2006; 201:7-14.
Takagi K, Okuda-Ashitaka E, Ito S et al. Involvement of stem cell factor and its receptor tyrosine kinase c-kit in pain regulation. Neurosci 2008;153:
Any papers are now emerging showing mechanisms of pain relief.
Veterinary Regenerative Medicine 101

21. Pain Relief Mechanisms
12/13/12
Model – ligation of masseter muscle nerve (constriction injury)
Normal
Highly Sensitized
Von Frey filament – larger = bigger filament needed to elicit a withdrawal
Treatment – MSC low 1500, High 1.5 M IV tail vein
Measure hypersensitive - Opioid (mu) receptor agonist
Guo et al, Stem Cells 2011;29(8):
Veterinary Regenerative Medicine 101

22. Anti-inflammation / Anti-fibrosis
12/13/12
Co-staining of IL1-RA (red) protein and subpopulation of MSCs (DAPI, blue).
Ortiz et al, PNAS 2007.
(Tulane Univ)
Interleukin 1 is the really bad actor in the joint inflammation and pain and OA. This study by Darwin Prokop’s Tulane group showed that the MSC can produce IRAP, the blocking protein to IL-1. This slide shows co-staining of a stem cell (DAPI stain blue) and IL1-RA (red).
Veterinary Regenerative Medicine 101

23. Mechanisms of Regeneration
12/13/12
Differentiation into tissue
Nerve
Bone
Cartilage
Liver
Fat-derived
Stem Cells
Cardiac
Fat
Angiogensis/
Anti-apoptosis
Gene Therapy
Muscle
(Photo courtesy Cytori Therapeutics)
Reviewed in: Tobita M. Adipose-derived stem cells: current findings and future perspectives. Disc Med 2011;11(57):
Veterinary Regenerative Medicine 101

24. Mechanisms of Regeneration
12/13/12
Stimulation of MSC Proliferation
This is an in vitro experiment to show that various stem cell types have one effect by stimulating local stem cells to proliferate
Kol et al (UCD). EVJ 2012.
Veterinary Regenerative Medicine 101

25. Mechanisms of Regeneration
12/13/12
Stimulation of MSC Migration
This is an in vitro experiment to show that various stem cell types have one effect by stimulating local stem cells to proliferate
Kol et al (UCD). EVJ 2012.
Veterinary Regenerative Medicine 101

26. Cartilage Regeneration Model
12/13/12
Adipose stem cell therapy of full thickness defects. Single treatment. A common goal would be to repair damaged cartilage. In this model, Dragoo et al at Stanford U showed that they could repair a full-thickness cartilage injury in the rabbit knee joint. They created a 3 x 4mm deep defect to subchondral bone in the lateral femoral condyle. Either fibrin (control) or fibrin + ASCs were implanted into the defects and then the rabbits were followed 8 weeks. As you can see, the 8 week repair was substantially thicker and more complete in the ASC group. 100% of treated and 8% of controls showed defect healing and successful integration into surrounding native cartilage. Not a dog, but a good first look.
At 8 weeks, 12/12 (100%) of defects in treated group healing with hyaline-like cartilage. Only 1/12 (8%) of controls healed.
Dragoo J et al, “Healing full-thickness cartilage defects using adipose-derived stem cells” Tiss Eng 2007;13(7): (Stanford)
Veterinary Regenerative Medicine 101

27. Anti-apoptosis Mechanism
12/13/12
Here is one clear example of homing ability and of anti-apoptosis of the MSC and ability to rescue dying cells. This is a model of stroke (infarct) of brain in a rat. After the infarct is created, adipose stem cells are injected intravenously up to 124 hours after the infarct. It is clear to see the large stroke infarct in the control group and the much smaller injury in the treated group, as well as improving neurological recovery.
Untreated Control ADSC IA Treated Group
Leu et al, J Translational Med 2010;8(63).
Veterinary Regenerative Medicine 101

28. Anti-fibrosis Mechanism
12/13/12
Blue = fibrosis
In this model of liver fibrosis in the mouse, adipose stem cells prevented fibrosos and increased the anti-fibrogenic cuytokine, IL-10
Control MSC
Mouse Liver Fibrosis – CCl4 – BM-MSC IV Infusions
Decrease TGF-B (decrease response of stellate cells)
Increase IL-10 (antifibrogenic cytokine)
Fang et al, Transplantation 78:1;2004
Veterinary Regenerative Medicine 101

29. Homing and Angiogenesis
12/13/12
Ischemia Model – Adipose Cell Therapy
7 days post ischemia - IV
Saline Control
Adipose Stem
Cell Treated
In this model, the femoral artery AND vein were ligated and adipose stem cells were given IV in the tail vein. By day 7, most of the ligated vessel limb was reperfused and the controls did not reperfuse (red = perfusion and blue = poor perfusion on doppler)
Miranville, Circulation, 2004
Laser Doppler Blood Flow
Veterinary Regenerative Medicine 101

30. Influenced by injury micro-environment
Roles / Functions
12/13/12
“Stem cells are injury-specific, perfectly choreographed pharmaceutical factories”
Influenced by injury micro-environment
“Paramedics”
Dr. Arnold Caplan, Case Western Reserve University
Veterinary Regenerative Medicine 101

31. ‘Activation’ of Stem Cells
12/13/12
Annu. Rev. Pathol. Mech. Dis :457–78
“In vivo use of hMSCs for therapeutic indications does not require priming of MSCs.”
Although stem cell activation is being promoted commercially, the data says that with MSCs that activation is not needed.
Veterinary Regenerative Medicine 101

32. Summary - Regenerative Medicine
12/13/12
1. Goal of Regenerative Medicine is to return damaged tissue to normal state.
2. Regenerative cells function by:
- Homing
- Differentiation into needed tissues
- Trophic stimulation of regeneration
3. Activation of stem cells is not necessary for therapeutic effects, and may be harmful.
Veterinary Regenerative Medicine 101