6 th International Conference on Neonatal Stem Cell-Based Therapy in Pulmonary & Childhood Pulmonary Vascular Vascular Disease Disease Stella Kourembanas, MD Harvard Division of Newborn Medicine � No Disclosures : 6 th International Conference on Neonatal & Childhood Pulmonary Vascular Disease Harvard Medical School Boston Children’s Hospital Bone Marrow-Derived Stem Cells Outline � Hematopoeitic stem cells (HSCs) 1. Mesenchymal Stem Cell-based therapies for diseases of the lung � Endothelial progenitor cells (EPCs) Bronchopulmonary Dysplasia (BPD) � Pulmonary Hypertension (PH) � � Mesenchymal stromal (stem) cells or bone marrow stromal cells (MSCs, BMSCs) 2. Mechanism of Mesenchymal Stem Cell action 1
MSC Differentiation Mesenchymal Stromal (Stem) Cells � Derived from bone marrow (BMSC); blood; and other tissues � Immunodepletion by FACS Analysis Using MSCs ISCT Guidelines � Can differentiate into connective tissue lineages Teeth Therapeutic Effects of MSCs Preclinical & Clinical Uses of MSCs � Clinical trials on autologous and allogeneic � Animal models of lung injury MSCs, including � IPF, ALI, BPD � Crohn’s, Multiple Sclerosis, End stage liver � Animal models of Pulmonary Hypertension disease, GVHD, ventricular dysfunction, MI, refractory SLE, Diabetes, COPD � Prochymal TM , commercial MSC prep (Osiris Therapeutics Inc, Columbia, MD) � FDA approved for phase III trial for Crohn’s Disease http://clinicaltrials.gov 2
Bronchopulmonary Dysplasia Mesenchymal Stem Cells for Lung (BPD) Injury Hyperoxia-induced neonatal lung injury model: BPD � Prolonged lung inflammation � Alveolar simplification; thickened alveolar septae; & dysmorphic vascular growth � PH (RVH and lung vascular remodeling) BPD BMSC-CM (cell-free) but NOT BMSCs MSCs and MSC-Conditioned Media (CM) Prevent Alveolar Injury Prevent PH in Hyperoxia-BPD Model 75% Oxygen 21% Oxygen Cells PASMC BMSC Media Aslam et al., Am J Respir Crit Care Med. 2009;180:1122-30 3
Late MSC-CM Treatment Reduces Lung BPD Rescue: Experimental Design Inflammation and Reverses RVH in BPD Model Macrophages/BAL Fulton’s Index (RVH) * 800000.0 0.5 * 0.4 600000.0 # 0.3 400000.0 0.2 200000.0 0.1 0.0 0.0 (BAL cell count) (CT Angio) MSC-CM MLF-CM Normoxia MSC-CM MLF-CM Normoxia 75% Oxygen 75% Oxygen MSC-CM Reverse PA Pruning MSC-CM Normalize Long-Term Lung in Hyperoxia-BPD Model Function in Hyperoxia-BPD Model Hansmann, G., Fernandez-Gonzalez, A., et al., Pulm. Circ. 2012;2:170-81 Hansmann, G., Fernandez-Gonzalez, A., et al., Pulm. Circ. 2012;2:170-81 4
Mesenchymal Stem Cells & MSCs & MSC-CM Treatment of PH– Hypoxic Mouse Model Experimental BPD � Prevent lung injury, inflammation, and PH � Effect of MSCs on reversing � Reverse lung fibrosis and PA devascularization established PH? � Partially reverse alveolar injury � Normalized long term lung function � Gene delivery in vivo : Heme � Reversed PH and RVH Oxygenase-1 MSC-CM are more effective than MSCs on preventing and reversing lung disease Enzymatic Activity of Heme Experimental Design Oxygenase (HO) Adult WT and H0-1 KO Mice Inflammation in hypoxia (8.5 % O 2 ) 0 CO Guanylyl cyclase NADPH Platelets HO-1,2 Heme Fe Fibrinolysis GTP cGMP O Biliverdin-IX H01-MSCs 2 IV week WT-MSCs 5 Biliverdin reductase injection Vascular smooth muscle cell PBS or MLFs or PASMCs Bilirubin-IX Relaxation week 7 Functional and morphological analysis: Antioxidant RVSP, RV/LV+S and PA remodeling 5
HO-1 -MSCs Reverse Hypoxic PH H0-1- MSC Transplantation Prevents RV Failure/Thrombus in Hypoxic HO-1 Null Mice RVSP (mm Hg) * (8) (8) Normal RVSP in H0-1 -MSC- (10) (13) HO-1 +/- HO-1 -/- RV injury Treated Hypoxic WT Mice Normoxia Animals with thrombus (%) 70 60 0.4 50 § 40 RV/LV+S 0.3 30 * (8) 20 Normal RV Weight in H0-1 - (8) 7 wks 0.2 ** 10 (10) (13) MSC-Treated Hypoxic WT Mice Hypoxia 0 0.1 H0-1 -MSC PBS 0 Liang et al., Stem Cells 2011; 29:99-107 Normoxia PBS SHO1- WT-FVB- Liang et al., Stem Cells 2011; 29:99-107 Hypoxia x 7 weeks Control BMSC BMSC Rapid Clearance of Donor MSCs Mechanisms of MSC Action: Donor MSC per 10 6 Recipient Lung Cells 3500 Normoxia Hypoxia 3000 1. Transdifferentiation into lung cells PBS Control 2500 2. Stimulation of endogenous lung 2000 progenitors cells to repair lung injury 1500 3. Paracrine:Immunomodulation;anti- 1000 inflammatory effects 500 0 0 2 4 6 8 10 12 14 16 Days post injection 6
MSC-CM Increase Endogenous Lung Transplanted MSCs express HO-1 Progenitor Cell Number In Vivo hHO1 mRNA per resident MSC 120 1 Hrxa + PASMC-CM 0.9 Ave No. BASC/TB 100 0.8 * Hrxa + MSC (arbitrary units) 0.7 Hrxa + MSC-CM 80 0.6 0.5 * 60 0.4 0.3 40 0.2 0.1 20 0 Bronchioalveolar stem cells Specimen Groups 0 (BASCs) Day 1 Day 2 Day 7 Day 14 Tropea et al., Am J Physiol Lung Cell Mol Physiol. 2012;302:L829-37. Mechanisms of MSC Action: Conditioned Media Proteomics Immunomodulation Among Proteins Detected: Respiratory Disease � CD63, CD81, moesin, Immune Response & Inflammation HSP90, HSP 70 HO-1 Cell-Cell Interactions & Cell Motility Proliferation & CO Apoptosis 0% 10% 20% 30% 40% 50% 60% Fraction of BMSC-CM Proteome Modified from: Iyer & Rojas, Expert Opinion on Biological Therapy, 2007 7
Exosome Release by MSCs Size Fractionation of MSC-CM Multivesicular Body and EM Analysis MSC Exosome Paracrine Factors Recipient Cell Conventional view of paracrine secretion of soluble proteins Recipient Cell Exosomes as mediators of MEX: MSC-derived exosomes paracrine effect FEX: Fibroblast- derived exosomes Modified from Lai et al, Regen Med 2011 Lee et al., Circulation. 2012 ;126:2601-11. MEX Mediate the Anti-inflammatory Experimental Design Effects of MSCs on the Lung Hypoxia Inflammation MEX FEX Vascular Remodeling RV Systolic Pressure Injection, JV or TV RV Hypertrophy 2, 4, 7 days, 3 weeks x 48 hrs x 48 hrs Lee et al., Circulation. 2012 ;126:2601-11. 8
Hypoxia Induces an Early Peak of Dose-Dependent Effects of MEX Lung Inflammation in Mice on Hypoxic Lung Inflammation (I) BAL Macrophage # (x 10 5 / ml) 10 MEX MEX 9 Low dose Low dose * 8 7 6 5 4 3 2 1 0 Days in Hypoxia 0 2d 4d 7d 9d 11d 3w Hypoxia Vergadi et al., Circulation. 2011;123:1986-95. Lee et al., Circulation. 2012 ;126:2601-11. Dose-Dependent Effects of MEX Systemic Infusion of MEX Inhibits on Hypoxic Lung Inflammation (II) Chronic HPH MEX MEX High dose Low dose High dose Alveolar M Φ s (x10 3 ) Hypoxia, 7 days Days in Hypoxia x 3 weeks Lee et al., Circulation. 2012 ;126:2601-11. 9
Systemic Infusion of MEX Inhibits MEX Inhibit PASMC Proliferation Hypoxic Vascular Remodeling ns 1.2 Relative Proliferation 1 * * 0.8 0.6 * 0.4 0.2 0 FBS (5%) - + + + + + mMEX - - 125 62.5 31.5 16 (ng/ml) For 3 weeks Human & Mouse MEX Suppress the Hypoxic Activation of STAT-3 In Vivo Lung Cultured hPAECs Lee et al., Circulation. 2012 ;126:2601-11. 10
MEX Treatment Modulates Regulation of Hypoxic Lung Signaling Pathways by MSC-derived Exosomes (MEX) Lung miR-204 Levels ** ** Relative levels of miRNA-204 Hypoxia 48 h Acknowledgments Summary � Mesenchymal stem cells � Changjin Lee � S. Alex Mitsialis � Biomarkers of health and disease � Kostis Sdrimas � Georg Hansmann � Xianlan Liu � Therapeutic agents in lung/CVR disease; Gene delivery � Angeles Fernandez- � Helen Christou Gonzalez � Mechanism of action � Tom Martin � Sally Vitali � Mark Perrella � Olin Liang � Paracrine Release of anti-inflammatory cytokines � Muhammad Aslam � Carla Kim � Kristen Tropea-Leman � Georgios Konstantinou � Rajiv Baveja Exosomes Structural & Functional Repair [Immunomodulation; mRNA, miRNA, proteins, resident progenitor cell stimulation] etc. 11
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