Slide 1

The ConceptsVet-Stem Credentialing CourseVeterinary Regenerative Medicine 101

Slide 2

Evidence of potential mechanisms Evidence of efficacy Evidence of safety Formulary for how to use in practice

Regenerative Medicine

What should you ask about any new therapy?

Slide 3

VRM 101 The ConceptsVRM 102 Evidence-Based Regenerative Medicine –

Pain and Orthopedic UseVRM 103 Stem Cell Mediated RegenerationVRM 104A Small Animal Clinician’s ApproachVRM 104B Equine Clinician’s ApproachVRM 105A Small Animal Adipose Harvest / InjectionVRM 105B Equine Adipose Harvest / Injection

Course Outline

Slide 4

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

Regenerative Medicine

Module Outline

Slide 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 IntegrumRestore to Original

Slide 6

Regenerative MedicineDamage Repair

Regenerate

Slide 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

Slide 8

What are Stem Cells?

Pericytes on blood vessels. Courtesy Arnold Caplan and

Bruno Peault

Stem Cells are:

– Primitive cells present in almost every tissue

Slide 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.

Slide 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.

Slide 11

What are Stem Cells?

Dividing stem cells. Courtesy Salk Institute.

Stem Cells are:

– Primitive cells present in almost every tissue

– Able to become different types of tissue:

Tendon, Ligament, Bone

– Self-renewing

Slide 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

Slide 13

Definitions

Multipotent turn into any cell line of same germ layerPluripotent turn into any cell line except placentalTotipotent turn into any cell type including placental

Autograft from animal A, into animal AAllograft from animal B, into animal AXenograft from species B, into species A

Mesenchymal originating from mesoderm

Slide 14

A Rose By Any Other Name

Stem cells aka…

Mesenchymal stem cellsMesenchymal Stromal cellsMultipotent / Pluripotent cellsStromal vascular fractionNucleated fraction

Slide 15

Embryonic vs Adult Stem Cells

Embryonic 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

Gruen L and Grabel L, Concise Review: “Scientific and Ethical Roadblocks to Human Embryonic Stem Cell Therapy.” Stem Cell 2006;24;2162-2169.

Slide 16

How to Use Stem Cells

Caplan, J Cell. Physiol. 2007, 213: 341-347

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

Slide 17

Stem Cell Mediated Regeneration

1. Homing (like WBC)

2. Differentiationa. Direct differentiation into needed cell types

b. Recruit and stimulate mitosis of local progenitor cells

3. Trophic support - growth factors and cytokinesa. Block pain (opioid receptor agonist)

b. Down-regulate inflammatory mediators

c. Block cell death (anti-apoptosis)

d. Stimulate angiogenesis

e. Anti-fibrosis (block scar)

Slide 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)

Slide 19

Cruciate Ligament - Chondroprotection

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

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.

Slide 20

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. Anesth Analg 2007;104:944-948. 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;153:1278-88.

Pain Relief Mechanisms

Slide 21

Model – ligation of masseter muscle nerve (constriction injury)

Pain Relief Mechanisms

Guo et al, Stem Cells 2011;29(8):1294-1303.

Normal

Highly Sensitized

Slide 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)

Slide 23

Mechanisms of Regeneration

Differentiation into tissue

(Photo courtesy Cytori Therapeutics)

Nerve

Bone

Cartilage

Liver

Fat-derivedStem Cells

Cardiac

Fat

Angiogensis/Anti-apoptosis

Gene Therapy

Muscle

Reviewed in: Tobita M. Adipose-derived stem cells: current findings and future perspectives. Disc Med 2011;11(57):160-170.

Slide 24

Mechanisms of Regeneration

Stimulation of MSC Proliferation

Kol et al (UCD). EVJ 2012.

Slide 25

Mechanisms of Regeneration

Stimulation of MSC Migration

Kol et al (UCD). EVJ 2012.

Slide 26

Cartilage Regeneration Model

Dragoo J et al, “Healing full-thickness cartilage defects using adipose-derived stem cells” Tiss Eng 2007;13(7):1615-21. (Stanford)

At 8 weeks, 12/12 (100%) of defects in treated group healing with hyaline-like cartilage. Only 1/12 (8%) of controls healed.

Slide 27

Anti-apoptosis Mechanism

Untreated Control ADSC IA Treated Group

Leu et al, J Translational Med 2010;8(63).

Slide 28

Anti-fibrosis Mechanism

Mouse Liver Fibrosis – CCl4 – BM-MSC IV Infusions1. Decrease TGF-B (decrease response of stellate cells)2. Increase IL-10 (antifibrogenic cytokine)

Fang et al, Transplantation 78:1;2004

Control MSC

Blue = fibrosis

Slide 29

Homing and Angiogenesis

Ischemia Model – Adipose Cell Therapy7 days post ischemia - IV

Laser Doppler Blood Flow

Saline Control

Adipose StemCell Treated

Miranville, Circulation, 2004

Slide 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

Slide 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

Slide 32

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.

Summary - Regenerative Medicine