The Concepts Vet-Stem Credentialing Course Veterinary Regenerative Medicine 101

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

2. Regenerative Medicine 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

3. Course Outline 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

4. Regenerative Medicine 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

5. What is Regenerative Medicine? 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

6. Regenerative Medicine Damage Repair Regenerate

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. What are Stem Cells? Stem Cells are: • Primitive cells present in almost every tissue Pericytes on blood vessels. Courtesy Arnold Caplan and Bruno Peault

9. What are Stem Cells? Stem Cells are: • Primitive cells present in almost every tissue • Able to become different types of tissue: Tendon, Ligament, Bone Stem cells differentiated into cardiomyocytes using growth factors. Courtesy NIH.

10. What are Stem Cells? Stem Cells are: • Primitive cells present in almost every tissue • Able to become different types of tissue: Tendon, Ligament, Bone Stem cells differentiated into cardiomyocytes using growth factors. Courtesy NIH.

11. What are Stem Cells? Stem Cells are: • Primitive cells present in almost every tissue • Able to become different types of tissue: Tendon, Ligament, Bone • Self-renewing Dividing stem cells. Courtesy Salk Institute.

12. What are Stem Cells? Stem Cells are: • Primitive cells present in almost every tissue • Able to become different types of tissue: Tendon, Ligament, Bone • Self-renewing • Pharmaceutical Factories

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. 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 Source: early embryo ethical dilemma Differentiate into all tissues Purpose: form organism Form Teratomas UNPREDICTABLE Embryonic vs Adult Stem Cells Adult Source: all adult tissues (?) no ethical dilemma Differentiate into most tissues Purpose: Regenerate No evidence of Teratoma formation Gruen L and Grabel L, Concise Review: “Scientific and Ethical Roadblocks to Human Embryonic Stem Cell Therapy.” Stem Cell 2006;24;2162-2169.

16. 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 How to Use Stem Cells Caplan, J Cell. Physiol. 2007, 213: 341-347

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

18. Homing Mechanism – Fracture Model Homing of luminescent adipose stem cells to fracture site from IV administration. S-W Lee et al, J Ortho Res, 2009 (Stanford Univ)

19. Cruciate Ligament - Chondroprotection 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. Toghraie et al, “Treatment of osteoarthritis with infrapatellar fat pad derived mesenchymal stem cells in rabbit”The Knee 2011;1:71-75.

20. Pain Relief Mechanisms • Eaton M. Cell and molecular approaches to the attenuation of pain after spinal cord injury. J Neurotrauma 2006;23(23/4):549-59. • Guo – Bone marrow stromal cells produce long-term pain relief in rat models of persistent pain. Stem Cells 2011;29(8):1294-1303. • Klass M, Gavrikov V, Csete M et al. Intravenous mononuclear marrow cells reverse neuropathic pain from experimental mononeuropathy. AnesthAnalg2007;104:944-948. • Malik RA, Veves A, Tesfaye S. Ameliorating human diabetic neuropathy: Lessons from implanting hematopoietic mononuclear cells. ExperNeuro2006; 201:7-14. • Takagi K, Okuda-Ashitaka E, Ito S et al. Involvement of stem cell factor and its receptor tyrosine kinasec-kit in pain regulation. Neurosci 2008;153:1278-88.

21. Pain Relief Mechanisms Model – ligation of masseter muscle nerve (constriction injury) Normal Highly Sensitized Guo et al, Stem Cells 2011;29(8):1294-1303.

22. Anti-inflammation / Anti-fibrosis Co-staining of IL1-RA (red) protein and subpopulation of MSCs (DAPI, blue). Ortiz et al, PNAS 2007. (Tulane Univ)

23. Mechanisms of Regeneration Differentiation into tissue Muscle Liver Cardiac Angiogensis/ Anti-apoptosis Nerve Fat-derived Stem Cells Bone Fat Cartilage Gene Therapy (Photo courtesy Cytori Therapeutics) Reviewed in: Tobita M. Adipose-derived stem cells: current findings and future perspectives. Disc Med 2011;11(57):160-170.

24. Mechanisms of Regeneration Stimulation of MSC Proliferation Kol et al (UCD). EVJ 2012.

25. Mechanisms of Regeneration Stimulation of MSC Migration Kol et al (UCD). EVJ 2012.

26. Cartilage Regeneration Model 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):1615-21. (Stanford)

27. Anti-apoptosis Mechanism Untreated Control ADSC IA Treated Group Leu et al,J Translational Med 2010;8(63).

28. Anti-fibrosis Mechanism Blue = fibrosis 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

29. Homing and Angiogenesis Ischemia Model – Adipose Cell Therapy 7 days post ischemia - IV Saline Control Adipose Stem Cell Treated Miranville, Circulation, 2004 Laser Doppler Blood Flow

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

31. ‘Activation’ of Stem Cells “In vivo use of hMSCs for therapeutic indications does not require priming of MSCs.” Annu. Rev. Pathol. Mech. Dis. 2011. 6:457–78

32. Summary - Regenerative Medicine 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.