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CELLULES SOUCHES MESENCHYMATEUSES ET MALADIES RESPIRATOIRES Master 2 BCPP

CELLULES SOUCHES MESENCHYMATEUSES ET MALADIES RESPIRATOIRES Master 2 BCPP Épithéliums structures d ’ interface Université Paris Diderot Christine Clerici U700 – CRB3 UFR de Médecine Paris Diderot. MSC A VISÉE THÉRAPEUTIQUE. MSC et greffe tissulaire : régénération des tissus

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CELLULES SOUCHES MESENCHYMATEUSES ET MALADIES RESPIRATOIRES Master 2 BCPP

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  1. CELLULES SOUCHES MESENCHYMATEUSES ET MALADIES RESPIRATOIRES Master 2 BCPP Épithéliums structures d’interface Université Paris Diderot Christine Clerici U700 – CRB3 UFR de Médecine Paris Diderot

  2. MSC A VISÉE THÉRAPEUTIQUE • MSC et greffe tissulaire : régénération des tissus • réparation cardiaque • ostéogénèse imparfaite • MSC et propriétés immuno-modulatrices • traitement de la GVH • insuffisance rénale • hépatite • diabète

  3. MSC A VISÉE THÉRAPEUTIQUE et PATHOLOGIES PULMONAIRES • MSC et greffe tissulaire : fusion ou repopulation? • Poumon sain (Krause, 2001) • Fibrose pulmonaire à la bléomycine (Ortiz, 2003; Kotton, 2004) • Poumon radique (Theise, 2002) • Broncho-dysplasie (van Haaften, 2009) • MSC et propriétés immuno-modulatrices • Fibrose pulmonaire induite par la bléomycine (Ortiz, 2007) • Fibrose pulmonaire induite par la silice (Lassance, 2009) • Broncho-dysplasie (van Haaften, 2009; Aslam, 2009) • MSC vecteurs thérapeutiques

  4. MSC et Syndrome de détresse respiratoire aiguë

  5. Les Traitements Pharmacologiques Ne Sont Pas Efficaces dans le SDRA • Glucocorticoids • Surfactant therapy • Lisofylline • Ketoconazole • Inhaled nitric oxide • Procysteine • Neutrophil elastase inhibitor • Activated Protein C

  6. Quels effets des MSC dans des modèles pré-cliniques de lésions inflammatoires alvéolaires aiguës ? 1. MSC et ALI dans des modèles animaux Effets de CSM in vivo dans un modèle d’oedème alvéolaire induit par l’endotoxine chez la souris Effets des CSM in vivo dans un modèle de VILI chez la souris Effets des CSM in vivo dans un modèle de pneumopathie bactérienne 2. MSC et ALI dans un modèle de poumon humain : études in vitro et in vivo

  7. PBS (n = 31) MSC (n = 30) EFFECT OF MSC ON SURVIVAL AND LUNG HISTOLOGY IN MICE AFTER LPS E. COLI +24/48H 0 +4H E.Coli IT MSC IT prélèvements 100 ** 75 50 48 h Survival (%) 25 **p < 0.01 0 0 10 20 30 40 50 Hours GUPTA ET AL, J IMMUNOLOGY, 2007

  8. MSC REDUCES PULMONARY EDEMA AND BAL PROTEIN IN MICE AFTER LPS 250 5 200 4 ** 150 3 Excess Lung Water (µl) BAL Protein (mg/L) ** 100 2 50 1 0 0 PBS MSC MSC PBS **p=0.005 **p=0.001

  9. B A 300 100 250 * 75 200 * 48 h Excess Lung Water (µl) 48 h Survival (%) 50 150 PBS (n = 13) 100 25 MSC (n = 11) 50 0 0 10 20 30 40 50 0 hours PBS MSC EFFECT OF MSC ON SURVIVAL AND PULMONARY EDEMA IN MICE AFTER ESTABLISHMENT OF E. COLI PNEUMONIA +24/48H 0 +4H E.Coli 106CFU IT MSC IT prélèvements Gupta et al, unpublished data, 2009

  10. EFFECT OF MSC ON BACTERIA GROWTH IN MICE AFTER ESTABLISHMENT OF E. COLI PNEUMONIA Krasnodembskaya, Stem Cells, 2010

  11. MSC THERAPY ENHANCES THE RESOLUTION OF STRUCTURAL LUNG INJURY FOLLOWING VILI.

  12. MSC THERAPY ENHANCES THE RESOLUTION OF STRUCTURAL LUNG INJURY FOLLOWING VILI. Curley G F et al. Thorax 2012;67:496-501

  13. MSC THERAPY MODULATES THE INFLAMMATORY RESPONSE TO VILI. Curley G F et al. Thorax 2012;67:496-501

  14. EFFECT OF MSC ON HUMAN LUNG INJURY INDUCED BY ENDOTOXIN Lee JW et al, PNAS, 2009

  15. EFFECT OF MSC ON HUMAN LUNG INJURY INDUCED BY ENDOTOXIN - Right or left lung selected, surgical preparation - Begin perfusion without blood 6 Instill 5 x 10 allogeneic human mesenchymal stem cells or MSC conditioned medium to RML • Measure AFC over 1 h • in RUL (Control) • Measure AFC over 1 h • in RML (endotoxin) 1 h 1 h 4 h 1 h • If AFC >10%/h in RUL, • Add 100 ml Fresh Whole Blood to Perfusate • Instill 0.1 mg/kg of endotoxin into the airspaces • of the RML • - Lung Temp 36°C • Apply 10 cm H20 CPAP • (95% O2 & 5% CO2 )

  16. 6 6 6 6 8 ± 5 x 10 cells 13 ± 11 x 10 cells 6 ± 5 x 10 cells 25 ± 25 x 10 cells Control Lung Lobe LPS Lung Lobe LPS + MSC Lung Lobe LPS + MSC CM Lung Lobe Absolute Neutrophil Counts P < 0.003 Lee, PNAS, 2009

  17. EFFECT OF MSC ON ENDOTHELIAL PERMEABILITY AND PULMONARY EDEMA IN HUMAN LUNG INJURED BY LPS A B 15 * 9 * 10 † Endothelial Permeability (%) Wed/Dry Ratio 6 † * † † 5 3 0 LPS + MSC Control LPS LPS + MSC CM Control LPS LPS + MSC LPS + MSC CM

  18. HYPOTHESIS FOR MSC IMPROVED ACUTE LUNG INJURY IN MICE OR HUMAN LUNGS No, < 5%, Gupta et al., 2007 • Engraftment • Immunomodulation • Alveolar fluid clearance • Lung protein permeability • Antibacterial properties

  19. HYPOTHESIS FOR MSC IMPROVED ALI IN MICE : IMMUNOMODULATION • No cell contact : MSC secretion of soluble factors – modulation by the inflammatory environment? • Cell-cell contact : Interaction with other inflammatory cells, leukocytes, macrophages…. • Candidate mediators released or induced by MSC • TGF-b • TNF-a induced protein (TSG 6) • PGE2 • IL-10 • IL-1ra …..

  20. MSC IMPROVED ALI IN MICE: IMMUNOMODULATION ALI - endotoxin ALI Pneumonia E. Coli 7000 4000 6000 3500 5000 3000 2500 4000 TNF-α BAL (pg/ml) MIP-2 BAL (pg/ml) 2000 3000 ** 1500 2000 1000 1000 500 0 0 MSC PBS MSC PBS Krasnodembskaya, 2010 Gupta et al., 2007

  21. MSC Upregulate Production of anti-inflammatory cytokines inEndotoxin Induced Lung Injury in Mice * 800 40 600 30 8 h BAL IL-10 (pg/ml) 24 h BAL IL-10 (pg/ml) 400 20 10 200 0 0 PBS MSC PBS MSC * 9000 7500 6000 24 h Plasma IL-10 (pg/ml) 4500 3000 1500 0 PBS MSC

  22. Nemeth et al, Nat Med, 2009 (ALI dans un modèle de sepsis par LC)

  23. MSC IMPROVED ALI IN MICE: IMMODULATION BY CELL-CELL INTERACTION Nemeth et al. Nat Med,2009

  24. HYPOTHESIS FOR MSC IMPROVED ACUTE LUNG INJURY IN MICE OR HUMAN LUNGS • Engraftment • Immunomodulation +++ • Alveolar fluid clearance • Lung protein permeability • Antibacterial properties

  25. 30 20 10 0 MSC IMPROVED ACUTE LUNG INJURY IN HUMAN LUNG: ALVEOLAR FLUID CLEARANCE † † Alveolar Fluid Clearance (%/h) * * MSC CM Control Normal Lung Fibroblasts MSC * P<0.0001 vs. Control LPS †P<0.0001 vs. LPS (0.1 mg/kg)

  26. MSC induced increase in Na-dependent AFC 30 20 * Alveolar Fluid Clearance (%/h) 10 0 + MSC CM + 5 x 10 M Amiloride + MSC CM -4 LPS Data as mean ± SD, * P < 0.05 Lee JW et al, PNAS, 2009

  27. ENaC Apical Membrane ENaC Total Cell Lysis † * † * * 85 kDa 64 kDa GAPDH † 120 * † 120 * 80 * 80 % of Control % of Control 40 40 0 0 Control + MSC Control +MSC Cytomix 50 ng/ml Cytomix 50 ng/ml

  28. Quels facteurs paracrines susceptibles d’améliorer la clairance alvéolaire ? • Diminution des cytokines pro-inflammatoires et augmentation des cytokines anti-inflammatoires dans le modèle ALI souris mais pas dans le modèle poumon humain. • Sécrétion de facteurs de croissance, KGF, angiopoietine-1 qui sont connus pour réduire les lésions pulmonaires dans les œdèmes lésionnels : bléomycine, VILI, hyperoxie, HCl, pneumopathie bactérienne.

  29. MSC sécrètent des facteurs de croissance KGF Angiopoietin-1 Control Control * 250 Cytomix +3%O2 Cytomix 200 * 150 concentration (pg/ml) concentration (pg/ml) 100 50 0

  30. MSC IMPROVED ALVEOLAR FLUID CLEARANCE IN HUMAN LUNG: ROLE OF KGF , # 30  20 Alveolar Fluid Clearance (%/hr) *,  10 * 0 Control LPS LPS + CM MSC + rhKGF (100ng) LPS + CM MSC (KGF siRNA) * P<0.001 vs. Control  P<0.03 vs. LPS (0.1 mg/kg) # P<0.01 vs. LPS + CM MSC (KGF siRNA)

  31. Mécanismes responsables de la restauration de la clairance alvéolaire par le KGF • Effet mitogène sur les cellules alvéolaires de type II, augmentant la capacité de transport de l’épithélium • Effet cytoprotecteur du KGF diminuant la perméabilité paracellulaire et/ou l’apoptose des cellules épithéliales • Le KGF pourrait augmenter la transcription des protéines impliquées dans le transport alvéolaire du sodium: ENaC & NaKATPase - 4. Le KGF pourrait augmenter l’adressage des canaux de type ENaC à la membrane apicale des cellules, un processus rapide déjà observé après stimulation de l’AMP cyclique en hypoxie (J Biol Chem, 2002)

  32. HYPOTHESIS FOR MSC IMPROVED ACUTE LUNG INJURY IN MICE OR HUMAN LUNGS • Engraftment • Immunomodulation +++ • Alveolar fluid clearance ++ • Lung protein permeability • Antibacterial properties

  33. MSC IMPROVED ACUTE LUNG INJURY : EPITHELIAL PERMEABILITY Upper compartment 131I-albumin Upper compartment lower compartment Human Alveolar Epithelial Type II Cells Lower compartment 0.4 M Microporous membrane Allogeneic Human Mesenchymal Stem Cells

  34. MSC IMPROVED ACUTE LUNG INJURY : EPITHELIAL PERMEABILITY A * * Permeability (%/24 h)  + MSC + Fibroblast Control Cytomix

  35. Absence d’effet du KGF sur la perméabilité épithéliale * * * *,  *,  Permeability (%/24 h) Cytomix 50 ng/ml + MSC KGF+Ang-1 Control + MSC (KGF siRNA) KGF

  36. 15 * ROLE OF ANGIOPOIETIN-1 * 10 Permeability (%/24 h)  # • Les MSC sécrètent l’angiopoietin-1 • Facteur de croissance vasculo-protecteur • MSC transfectées avec AP-1  diminution des lésions vasculaires et des lésions pulmonaires modèle ALI souris (Mei et al., 2008) 5 0 Control + MSC + MSC (Ang1 siRNA) + rhAng1 Cytomix 50 ng/ml Fang et al. , J. Biol. Chem., 2010, 285, 26211

  37. HYPOTHESIS FOR MSC IMPROVED ACUTE LUNG INJURY IN MICE OR HUMAN LUNGS • Engraftment -No • Immunomodulation -Yes • Alveolar fluid clearance -Yes • Lung protein permeability - Yes • Antibacterial properties ?

  38. MSC IMPROVED ACUTE LUNG INJURY IN MICE: ANTIBACTERIAL PROPERTIES 1. Les MSC ont-elles un pouvoir antimicrobien: nombre et croissance de bactéries in vitro 2. Les MSC permettent-elles de limiter la croissance et d’augmenter la clairance des bactéries dans un modèle in vivo de pneumonie bactérienne

  39. MSC REDUCES BACTERIA COLONY FORMATION in vitro

  40. MSC REDUCES E. Coli COLONY FORMATION in vitro  8 * 6 E. coli CFU (x 105/ml) 4 2 0 PBS MSC+ anti-LL37* Ab MSC Krasnodembskaya et al., Stem Cells, 2010

  41. Blood Bacterial Counts (CFU/ml) 100 4,000 hMSC n=34 * 75 3,000 50 3T3 n=27 Survival (%) 2,000 25 PBS n=42 1,000  0 0 12 24 36 48 0 3T3 (21) PBS (22) MSC (18) Intravenous hMSC Reduce Mortality & Bacteremia in Mice when Administered 4 hours after Peritoneal Pseudomonas Sepsis Time (h) MSC vs 3T3 < p 0.01 MSC vs PBS < p 0.01 MSC vs 3T3 p = 0.037, MSC vs PBS p = 0.005 Samarani et al, 2010

  42. 800 100 MSC 3T3 PBS ,√ P.aeruginosa in blood (x 102 cfu/ml) P.aeruginosa in blood (cfu/ml) 400 10 0 0 0 8 12 3T3 MSC PBS Time (h) Severity of Sepsis: the rise in bacterial growth in blood is prevented by MSC treatment MSC vs 3T3 p 0.0159 MSC vs PBS p 0.0161

  43. Neutralization of LL-37 Activity Abolishes the Antimicrobial Effect of MSC in vivo 105 106 104 105 E.coli CFU/ml in BAL *   E.coli CFU/ml in LH * 104 103 PBS +antiLL-37Ab + IgG MSC 103 102 +anti LL-37 Ab +IgG PBS MSC Krasnodembskaya et al., Stem Cells, 2010

  44. B A 50 100 ,√ 40 ,√ 30 Phagocytic Index Phagocytosis (%) 50 20 10 0 0 3T3 MSC PBS 3T3 MSC PBS C 4 3 E.coli cfu (x 106 /ml) in incubation medium ,√ 2 1 0 RPMI 3T3 MSC PBS MSC INCREASES PHAGOCYTIC ACTIVITY OF HOST IMMUNE CELLS Phagocytic activity of mouse peripheral blood monocytes

  45. Alternative Activated (Type II) Macrophages • Critical role in host defense for resolution of inflammation and with a high phagocytic activity against bacteria. • CD163 (haptoglobin-hemoglobin scavenger receptor) and CD206 (macrophage mannose receptor) are markers of type II macrophages. • Recent data suggest that CD163 can function as a macrophage receptor for gram positive and gram negative bacteria (Blood, 2009) • Contrast with TLRs which recognize soluble dissociated microbial components, not intact bacteria.

  46. Immunofluorescence for CD163 on the slides of mouse spleen MSC 3T3 PBS H&E staining of mouse spleen slides

  47. HYPOTHESIS FOR MSC IMPROVED ACUTE LUNG INJURY IN MICE OR HUMAN LUNGS • Engraftment -No • Immunomodulation – augmentation cytokines pro-inflammatoires • Alveolar fluid clearance – role du KGF • Lung protein permeability – role de l’angiopoietine • Antibacterial properties- peptides anti microbiens • Other mechanisms – microvesicules…..

  48. Therapie cellulaire dans le SDRA? • Confirmation que les MSC ont un effet bénéfique dans des pathologies respiratoires • Quels types cellulaires? • Quand les administrer et pendant combien de temps? • Quelle voie d’administration, IT, IV

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