Physiology and Applications of MSC Exosomes

1. Physiology and Applications of MSC Exosomes Douglas J. Spiel, MD

2. TOPICAL EXOSOMES

3. MSC Exosomes Arnold Caplan: • 2006 – “The trophic effects of MSCs may have profound clinical use.” • 2017 – “I now urge that we change the name of MSCs to Medicinal Signaling Cells …” • “It is, indeed, the patient’s own site-specific and tissue specific resident stem cells that construct the new tissue as stimulated by the bioactive factors secreted by the exogenously supplied MSCs” Revisionist Viewpoint!

4. 1917 – Julius Wagner-Jauregg • Patients with dementia paralytica after febrile illness frequently become lucid, sane. He began treating dementia paralytica patients with malaria for 8-12 febrile paroxysms then rescued with Quinine Sulfate and they became lucid. • 1927 – Nobel Prize in Medicine awarded to Julius Wagner-Jauregg for the healing properties of malarial fever • 1928 – Alexander Fleming discovers Penicillin • 1938 – Dr. Howard Florey reads Fleming’s paper. He is joined by Dr. Ernst Chain and Dr. Normal Heatley • 1942 – Mass production of Penicillin • WW I death rate from pneumonia: 18% • WW II death rate from pneumonia: <1%

5. Tomorrow’s Regenerative Medicine Textbooks • Bone Marrow Harvest MSCs Malaria • Adipose Harvest MSCs • Exosomes = Penicillin

6. What are MSC Exosomes? • Vesicles from 40-100 nm • Formed by a two-step budding process • Inward budding of membranous vesicles in a multivesicular-body • MVBs fuse with the plasma membrane to release their cargo • Transmembrane proteins are conserved! • Composed of lipids, proteins, mRNA, and miRNAs

7. I said transmembrane proteins are conserved from MSCs. Why? • MSCs are recruited at the site of injury by receptor-mediated interaction • MSC-derived vesicles bear the same membrane receptors of MSCs and it is likely for this reason that they accumulate at the site of injury by the same mechanism 4,5 • MSC exosomes via IV infusion were taken up by M2 Macrophages in spinal cord of injured rats

8. WHAT TYPES OF MEDICAL CONDITIONS MIGHT BE AIDED BY MSC EXOSOMES? • Musculoskeletal – Joints, Discs, Muscles, Bones, Ligaments, Tendons • Autoimmune – SLE (Lupus), RA, Dermatomyositis • Neurodegenerative – Multiple Sclerosis, Alzheimers, Parkinsons, Spinal Cord Injuries • Burns, Scars, Ulcers • Heart Disease – MI, CHF • Lung Disease – COPD, Pulmonary Fibrosis, Interstitial Lung Disease • Liver Disease • Kidney Disease • Inflammatory Bowel Disease – Ulcerative Colitis, Crohn’s Disease • Hair Loss

9. WHAT TYPES OF MEDICAL CONDITIONS MIGHT BE AIDED BY MSC EXOSOMES?(CONTINUED) • Cerebral Palsy, Autism • Neuropathy, CIDP • Erectile Dysfunction • Anorgasmia • Urinary Incontinence • Complex Regional Pain Syndrome • Chronic Pelvic Pain • Lyme Disease (Upregulate Macrophages) • Numerous Aesthetic Applications

10. What can Science teach us regarding Aging? By splicing the blood circulation of 2 animals together we have shown that young blood rejuvenates old tissues, bones, muscles, brain, and nerve tissue. Nik Spencer/Nature; Chart Data: A. Eggel & T. Wyss-Coray Swiss Med. Wkly 144, W13914 (2014) [Photograph]. Retrieved from https://www.nature.com/news/ageing-research-blood-to-blood-1.16762

11. What can Science teach us regarding aging? A Hong Kong billionaire backed a scientist from Standford (Wyss-Coray) to start Alkahest, Inc, in Menlo Park, California on 2014. To Treat Alzheimers they are sharing plasma from adults < 30 and transfusing it into Alzheimers patients.

12. What can Science teach us regarding aging? A second company, Ambrosia has started a similar trial in California, giving adults age 35+ transfusions from younger adults <25. This one-time infusion of a 2L bag of plasma (no blood cells) is offered for $8,000.

13. Why does plasma transfer work? Are we transfusing stem cells? No. Simply, there are not enough stem cells in blood/plasma. In fact, factors present in the plasma seem to upregulate the older stem cells themselves.

14. What might these factors be? EXOSOMES Mesenchymal Stem Cells [Photograph] Retrieved from http://stemcellorthopedic.com/mesenchymal-stem-cells/

15. Translational Studies Employing MSC-derived exosomes • TARGET TISSUES • Heart • Brain • Liver • Lung • Intestine • Skin/Wound • Limb Ischemia • Skeletal Muscles • Spinal Cord • Immune System These studies demonstrate that MSC-derived exosomes recapitulate the broad therapeutic effects previously attributed to MSCs via horizontal transfer of mRNAs, miRNAs, and proteins

16. Key Advantages of Exosomes: • Can travel via systemic therapy without risk of clumping • Can travel via local therapy • Cross the “Blood-Brain Barrier” • Deliver miRNA and mRNA • Can home • Not perceived as foreign • No first-pass lung removal as in MSCs • Can not transdifferentiate into other cells or into malignant cells • Easy to administer, store, freeze (Out of the box stem cell therapy!) • Easily controlled dosage • Potency related to age of parent MSC

17. Key Therapeutic Effects of MSC Exosomes • Influence Growth of Target Cells • Influence Phenotype • Contribute to Cell Fate Decision • Promote Regeneration • Immunomodulation • Anti-Inflammatory • Anti-Fibrotic

18. Some Key Immune and Growth Factors Present in MSC Exosomes • BMP-5 Stimulates bone growth • GDF-15 Growth Differentiation Factor-15 • OPG Stimulates bone growth/blocks osteoclast precursor formation • G-CSF Granulocyte Colony Stimulating Factor • SCF Stem Cell Factor shown to be responsible for stem cell and melanocyte growth • TGF-ß3 The most important anti-inflammatory protein. Converts inflammatory T-Cells to anti- inflammatory regulatory T cells. • VEGF Vascular Endothelial Growth Factor • ICAM-1 Binds inflammatory ligands on white cells

19. Some Key Immune and Growth Factors Present in MSC Exosomes • IL-1ra Binds and sequesters the inflammatory cytokine IL-1 • IL-6 Responsible for macrophage activation • IL-10 An anti-inflammatory cytokine responsible for immunomodulation and regulatory T cell conversion • MCP-1 Monocyte Chemoattractant Protein-1 is a chemokine that recruits mononuclear cells to the treatment area • MIP-1 Also known as CCL-4, this chemokine recruits mononuclear cells to the treatment area • PDGF-ßß Platelet Derived Growth Factor Beta

20. Some Key Immune and Growth Factors Present in MSC Exosomes • TIMP-1 Tissue Inhibitor of Matrix Metalloproteinases, blocks collagen and extracellular matrix degradation. Important for cartilage repair • TIMP-2 • HGF Hepatocyte Growth Factor • GDNF Glial-Derived Neurotrophic Factor • BDNF Brain-Derived Neurotrophic Factor • FGF Fibroblast Growth Factor • TNF-RI Binds and inactivates the inflammatory TNF-α • IGFR Insulin-like Growth Factor Receptor

21. Important Immunological Effects of MSC Exosomes • Downregulate TH1 and TH17 cells while upregulating TReg cells • Upregulate M2 macrophages at expense of M1 Macrophages

22. Stem Cells/ProductsWhat would be optimal?

23. Tissue Resident Stem Cells • Many human organs and tissues contain an undifferentiated population of tissue-resident cells facilitating repair and remodeling. • This population lies quiescently within the niche. • After activation, progenitor cells proliferate and migrate to sites of injury where they differentiate and acquire the mature phenotype.

24. Neural Stem Cells (NSCs) • - Subventricular zone • - Subgranular zone (hippocampal dentate nucleus) • Cells are quiescent within the neurogenic niche (ependymal cells, astrocytes, microglia, and blood vessels) • Astrocytes within the niche secrete VEGF and FGF-2 to promote maintenance. With aging, factors decline as does neurogenesis (altered microenvironment/signaling)

25. NSCs in Neurodegenerative Disorders • Alzheimer’s Disease • Amyloid-ß deposits (Extracellular) • Tau/Neurofibrillary Tangles (Intracellular) • Affects basal forebrain cholinergic neurons, hippocampus, and cortex. • Huntington’s Disease • Huntington protein • Damages GABAergic neurons in striatum and other cortical areas • Parkinson’s Disease • α-synuclein forms intracellularly in dopaminergic neurons of the midbrain. • NSCs are also involved in traumatic brain injury, stroke, Chronic Traumatic Encephalopathy, Multiple Sclerosis, and spinal cord injury

26. Skeletal Muscle Stem Cells Principle cell = Satellite cells Also non-satellite cells • Again, regenerative capability requires cooperation of multiple cell types. • Satellite cells are quiescent under normal physiological conditions. • Cues (such as exercise, injuries, disease) yields expansion and proliferation to form myoblasts or muscle. • Factors in the systemic environment of old animals mediate the conversion of satellite cells from myogenic to fibrogenic lineage. • In muscular dystrophies, myofibers are readily damaged and cell mediated repair leads to exhaustion of the satellite pool. • Satellite cells are markedly affected by the niche.

27. Liver Progenitor Cells (Multiple Types) Hepatoblasts • i.e.: Hepatic progenitor cells Cholangioblasts Regenerative ability requires cooperation between numerous cell types.

28. Skin-Derived Multipotent Stromal Cells • Progenitor cells from mesoderm and ectoderm

29. Lung Stromal and Progenitor Cells • Numerous different types of cells along the tracheobronchial tree

30. Cardiac Stem Cells • Niches in atria and ventricular apices. • Hypoxia favors quiescence • After myocardial infarction: • Decreased O2 in infarct area • Cardiomyocyte Necrosis • Death of resident stem cells • Scar yields anatomical barrier • Limited growth factors • Therefore difficult to repopulate after myocardial infarction

31. Migration of MSCs adjacent to the joint cavities crucial for chondrogenesis. • Joint resident MSCs are abundant and occupy several bone and joint cavity niches • chondrocyte progenitor cells (CPCs) • cartilage-derived stem/progenitor cells (CSPCs) • synovium-resident multipotent progenitor cells • osteoblasts/osteoclasts/resident MSCs within the subchondral bone • chondrogenic cells within the infrapatellar fat pad

32. Mechanism Underlying Exosome Mediated Cartilage Regeneration • Following injection, chondrocytes rapidly transported exosome intracellularly. • Highest dose was 10 µg/mL • Increased cartilage, extracellular matrix components (Collagen Type II, Glycosaminoglycans) increased expression levels of mRNA for cartilage matric protein (COMP) and chondrogenic factor TFG-ß1. • Increased M2 decreased M1 Macrophages in both cartilage and synovial tissues.

33. The balance of progenitor cell quiescence and activation is a hallmark of a functional niche and is regulated by internal and external signals leading to self-renewal and differentiation of progenitor cells.

34. Muscles, Capillaries, & Glucose Metabolism

35. Reduced capillarization (capillary rarefaction) contributes to aging by decreasing muscle mass, metabolism, fitness, and function by limiting delivery of Oxygen, amino acids, nutrients, and hormones. • Older adults have decreased capillarization compared with young adults. • Loss of muscle mass in aging = sarcopenia • Capillaries may interact with satellite cells • Satellite cell activation may be blunted by greater distance to capillaries/factors. • VEGF deficiency in mouse models shows a significant reduction in capillaries and muscle mass • 50% of adults greater than 65 years old show impaired glucose tolerance or Type II Diabetes Mellitus • Low Skeletal muscle capillarizationlikely contributes to impaired glucose tolerance and Type II Diabetes Mellitus

36. VEGF is essential for hypertrophy and angiogenesis • VEGF acts as a chemoattractant for macrophages which release IFT-1, and activates satellite cells • Increase in muscle mass = Increase in metabolism

37. Diabetic neuropathy affects 50% of patients with Type I and II Diabetes Mellitus • Both central and peripheral pathological process are at play • Hyperglycemia and resulting oxidative stress effect the local microenvironment of the spinal cord promoting activation of glial cells. • Activated microglia yield neuroinflammatory changes causing sensorial neuropathy • High glucose concentrations induce metabolic and enzymatic abnormalities disrupting the functions of mitochondria inducing the overproduction of ROS and RNS compared with oxidative stress. • A single MSC or MSC-CM treatment promoted re-establishment of redox homeostasis • MSC administration enhanced the levels of IC-10 and TGF-ß in the spinal cords of neuropathic mice

38. ExosomesIntra-articular exosomes Approximately 7 mg one week apart Approximately 7 mg • Assumption is that 1M MSC produces 80-150 µg or proteins • TGF-ß Peaked at 12 weeks • IL-10 Above baseline at 52 weeks • TNF-α – Trough at 12 weeks, below baseline at 52 weeks • GFR peaked at 12 weeks, above baseline at 52 wees. • Cr troughed at 12 weeks, below baseline at 52 weeks Biopsies at 3 months showed increased renal tubules

39. Mechanism of MSC Exosomes Mitigating Type-II DM and Impaired Glucose Tolerance • Anti-Inflammatory Effects • Anti-Inflammatory Effects • Decreased inflammation • M1 M2 • Th1 Th2 • Increased TReg • Increase anti-inflammatory cytokine (IL10, TGF-ß3, TIMP, TNFαRA, IL1RA) • Decreased inflammatory cytokines • Therefore better binding of insulin to receptors

40. Mechanism of MSC Exosomes Mitigating Type-II DM and Impaired Glucose Tolerance • Anti-Apoptotic Effects Decreased apoptosis of Beta cells in pancreas. Therefore increased Insulin production.

41. Mechanism of MSC Exosomes Mitigating Type-II DM and Impaired Glucose Tolerance • Pro-Growth Effects • Increased VEGF • Increased pro-angiogenic miRNAs • Increased formation of capillaries in muscle • Increased capillaries = increased surface area vessels: muscle • Increased activity of satellite cells • Increased muscle mass = Increased metabolism • Increased delivery of insulin by increased diffusible surface area • Therefore enhanced glucose flux from blood to muscle.

42. Key features of reperfusion injury after myocardial infarction: • Loss of ATP/NADH • Increased Oxidative Stress • Cell Death • MSC exosomes contain all 5 enzymes in the ATP-generating stage of glycolysis • GAPDH • PGK • PGM • ENO • PKm2

43. 30g, 10-12 week old mice (~1oz each) • Left coronary artery ligation for 30 minutes • 0.1-0.4 µg exosomes/mouse intravenously within tail vein 5 minutes prior to reperfusion. • Results: • Intact exosomes reduce myocardial infarct/reperfusion injury through direct interaction with myocardial cells • Exosomes prevent left ventricular dilation and improve cardiac function • Exosomes restore energy depletion nd redox state within 30 minutes • (ATP/ADP and NADH/NAD+ ratios increased) • Reduced oxidative stress • Exosome treatment recued inflammation after infarct/reperfusion decreased macrophage and neutrophil infiltration into heart. Decreased peripheral white blood cell count

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